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Fig. 2. Aberrant neuronal differentiation in Cux2 mutants. (A) Schematic of gene trap insertion mutation in intron 3 (in3) of murine Cux2/Cutl2 gene containing a promoterless neomycin (neo) selectable cassette splice acceptor (SA) and donor (SD) sites, and a long terminal repeat (LTR). (B) Western blot of two individual Cux2^+/+ (+/+), Cux2^neo/+ (+/-) and Cux2^neo/neo (-/-) E12.5 embryo lysates. Cux2 protein migrated near 110 kDa. Loading control westerns for the embryo lysates showed anti-alpha Tubulin levels migrate near 50 kDa. (C,D) Hematoxylin and Eosin staining of the neural tubes of E11.5 wild-type control (C) and Cux2^neo/neo mutant (D) embryos, which exhibit hypoplastic neural tubes and reductions in the mz, drg and ventral commissure (arrowhead). (E,J) Ki67 (green) and TuJ1 (red) staining in Cux2^neo/+ (E) and Cux2^neo/neo (J) E11.5 spinal cords. (F-L) P27^Kip1 staining in Cux2^neo/+ (F,G) and Cux2^neo/neo mutants (K,L) demonstrating reduced p27^Kip1 expression levels in the iz at E11.5. (G,L) High magnification of F,K, respectively, detailing p27^Kip1 activity in the iz (arrowhead). (J,M) Enlarged lumen (double-ended arrows) and hypoplastic drg (blue arrows). (H-N) Ki67 (green) and NeuN (red) expression in control Cux2^neo/+ (H,I) and Cux2^neo/neo mutant (M,N) spinal cords. (I,N) High magnification of motoneuron domains of E11.5 Cux2^neo/+ and Cux2^neo/neo spinal cords showing NeuN expression (red) in post-mitotic motoneurons (arrowhead). Scale bars: 250 µm in E; 500 µm in G.

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