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Fig. 3. Generation and testing of conditional Lhx1; Lhx5 double-knockout animals. (A) Simplified schematic adapted from Kwan and Behringer (Kwan and Behringer, 2002) and Zhao et al. (Zhao et al., 1999) showing the targeted alleles of Lhx1 and Lhx5 that were used in this study. (B) PCR amplification of a DKO embryo carrying the Nes^Cre;Lhx1^loxP and Lhx5^- alleles. The Cre transgene is detected as a 1 kb band. A 480-base-pair band is diagnostic for the Cre-deleted Lhx1^loxP allele. Neo is present in both Lhx5 heterozygotes and Lhx5 mutants. The absence of a wild-type Lhx5 band of approximately 430 base pairs distinguishes Lhx5 mutants from Lhx5 heterozygous animals. This wild-type Lhx5-specific band is absent in DKO embryos. (C,D) Lhx1-protein expression is nearly completely abolished in the DKO cord at E12.5, with the exception of a few ventral Lhx1⁺ escapees. (E) Expression of lacZ and NeuN in E11.5 spinal cords of Nestin^Cre;Rosa26^lacZ+/- animals. (F,G) High magnification of newly postmitotic NeuN⁺ interneurons (green) in E (box) showing colocalization with ß-gal (red; asterisk in G).

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