(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+/+ (+/+), Cux2neo/+ (+/−) and Cux2neo/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 Cux2neo/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 Cux2neo/+ (E) and Cux2neo/neo (J) E11.5 spinal cords. (F–L) P27Kip1 staining in Cux2neo/+ (F,G) and Cux2neo/neo mutants (K,L) demonstrating reduced p27Kip1 expression levels in the iz at E11.5. (G,L) High magnification of F,K, respectively, detailing p27Kip1 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 Cux2neo/+ (H,I) and Cux2neo/neo mutant (M,N) spinal cords. (I,N) High magnification of motoneuron domains of E11.5 Cux2neo/+ and Cux2neo/neo spinal cords showing NeuN expression (red) in post-mitotic motoneurons (arrowhead). Scale bars: 250 μm in E; 500 μm in G.