NCM controls expression of genes involved in bone resorption. (A) Using RT-qPCR to assay for Col1α1 mRNA as a marker for bone deposition shows a 2.5-fold increase in duck (dark blue) from HH34 (n=5) to HH37 (n=5) and 4-fold increase in quail (light blue) from HH34 (n=5) to HH37 (n=5). Levels of Col1α1 expression in chimeric quck (red) at HH34 (n=2) are like that observed in quail controls at HH37. (B) Levels of Mmp13 in duck show a 3-fold increase from HH34 to HH37, whereas quail have an approximately 35-fold increase in expression from HH34 to HH37. In chimeric quck at HH34, quail donor cells maintain their higher stage-specific and species-specific levels of expression with an approximately 37-fold increase in Mmp13. (C) There is no significant increase in Mmp9 expression from HH34 to HH37 in duck whereas quail have a 6-fold increase. In quck, there is a 4-fold increase in Mmp9 at HH34 like that observed in quail at HH37. P-values: quail at HH37 compared to HH34; duck at HH37 to HH34; and quck at HH34 to HH34. (D) Sagittal sections showing the dentary bone and Meckel’s cartilage in the lower jaw of quail at HH37. (E) Through in situ hybridization on adjacent sections and coincident with the intramembranous ossification of bone, Mmp13 transcripts can be observed in the osteoid matrix (yellow dashed line), but not in Meckel’s cartilage. (F) Mmp9 expression is localized to discrete domains in bone presumably coincident with the distribution of osteoclasts. Mmp9 is not detected in Meckel’s cartilage. (G) Endochondral ossification of cartilage is restricted to the proximal-most region of the duck lower jaw at HH39. (H, I) Mmp13 and Mmp9 are expressed in cartilage undergoing endochondral ossification. (J, K, N and O) Osteoid in quail and duck at HH36 and HH39. (L, M) In quck, duck host bone is Q¢PN-negative while quail donor bone is Q¢PN-positive (black cells). (P, Q, T and U) Mmp13 is expressed at higher levels in quail versus duck at HH36 and HH39. (R) Mmp13 is not detected on the duck host side of quck (S) but is highly expressed on the quail donor side coincident with Q¢PN-positive cells.