FIG. 4.

Reciprocal expression patterns of Hmgn1 and Sox9 during chondrogenesis in vitro in micromass cultures. (A) Schematic diagram of micromass culture method. Mesenchymal limb bud cells obtained from E10.5 embryos are dissociated by trypsin-collagenase treatment, collected and concentrated by centrifugation, and then resuspended in the culture medium at high density (2.0 × 10⁷ cells/ml). Cells are plated as a 10-μl spot and flooded with medium after 1 h of incubation and adhesion. (B) Detection of chondrocyte differentiation by Alcian blue staining. Mesenchymal cells plated at high density differentiate into chondrocytes after 3 to 5 days in culture. (C) Reciprocal expression of Hmgn1 and Sox9 transcripts during mesenchymal differentiation into nodules. Plus symbols indicate developing nodules. After 5 days, Hmgn1 expression is down-regulated in the fully differentiated region of the chondrogenic nodule, where Sox9 expression is strongly expressed. (D) Confocal immunofluorescence analysis of HMGN1 and SOX9 expression in 5-day-old differentiated nodules. (a) Low magnification. HMGN1 protein is absent from the chondrocytic nodule, while SOX9 protein is detected only within the nodule. (b) Higher magnification of the images shown in part a centered on a differentiating nodule. Note that in the DNA-Sox9 merge, the center of the nodule is mostly green (high Sox protein levels) and the surrounding cells are red (low Sox protein; DNA stain prevails), while in the DNA-HMGN1 confocal merge the center is red due to low HMGN1 (light green) and the surrounding cells are green due to relatively high levels of HMGN1. Proteins are shown in green, and DNA (Hoechst) is shown in red. Phase-contrast images are used to visualize node morphology. Scale bar, 50 μm.