Fig. 10. Analysis of the trunk neural crest cells in long-term culture. RT-PCR amplification of cranial crest-specific markers from cranial and trunk neural crest cultures. Id2 and Noelin are highly expressed in both cranial explants and 14-day-old cranial crest but not in the new trunk crest cultures (A). A histogram showing the number of cultures positive for Alcian Blue staining, i.e. undergoing chondrogenesis (B). Note that on the second week of incubation (D14), about 80% trunk neural crest cultures undergo chondrogenesis and RCAS::Hoxd10 infection can suppress chondrogenesis in these cultures. (C-J) In situ hybridization on trunk neural crest cultures incubated for 7 or 14 days. Hoxb4 (C) but not ColII (D) signal is detectable in most cells of the week-old trunk neural crest cultures. ColII expression (E,F) is detectable in chondrocytes present throughout the 2-week-old cultures. Double in situ hybridization (G-J) revealed that very none or few Hoxb4-expressing cells are also positive for ColII transcript. (K-W) Immunochemistry with antibodies revealing chondrocytes (COLII), smooth muscle cells (SMA), neuronal cells (NF200) and 3C2 antibody (RCAS-infected cells) demonstrates the effect of the ectopic trunk Hox expression on chondrogenesis in long-term trunk crest cultures. (K,O,T) Trunk neural crest culture grown for 7 days, analyzed for presence of chondrocytes (K), smooth muscle cells (O) and neurons (T). Note that no COL2-positive cells are detected in these cultures. (L,P,U) Similar cultures grown for 2 weeks have many chondrocytes that appear throughout the culture, whereas smooth muscle cells and neurons are still present. (M,R,V) Trunk neural crest cultures infected with RCAS::Hoxd10 virus and grown for 2 weeks display a much lower level of chondrogenesis with no effect on myogenesis and neurogenesis. (N,S,W) To ensure that the cultures were indeed expressing the RCAS construct, 3C2 antibody was used to reveal the extent of the retroviral infection.