Ectopic ossification and neurogenesis in neural crest cell (NCC)-specific integrin-linked kinase (ILK) knockout (NKO) mutant outflow tract (OFT). (A,B) Quantitative polymerase chain reaction (qPCR) verification of upregulated genes in NKO mutant OFT. (C-F’) β-Galactosidase (β-gal) and neurofilament staining of adjacent sections of control (C-D’) and NKO mutant samples; (E-F’) show reduced size of superior cervical ganglia (scg) and ectopic innervation/ganglia in NKO mutant OFT ((E,F), red arrowhead). Scale bar: 200 μm; ao aorta, cat common arterial trunk, pa pulmonary artery, vn vagal nerve. (G-J) Ectopic ossification in the CAT and OFT cushion of NKO mutant hearts at E12.5 revealed by Alcian blue (AB) staining (G,H) and in situ hybridization with a probe to Col1a2 (I,J). (K) Increased expression of Col1a2 in NKO mutant NCCs revealed by qPCR. (L) Increased bone morphogenetic protein 2K (BMP2K) expression and Smad1 phosphorylation in NKO mutant NCC relative to the controls revealed by western blot.