Fig. 10: Creb5 is necessary to maintain flat cells in the superficial zone of the articular cartilage in postnatal mice.

a Safranin O/Fast Green staining or RNAScope FISH of the articular surface of the knees of P21 Aggrecan1^{tm(IRES-CreERT2)}; Creb5^flox9/flox9 mice or Creb5^flox9/flox9 littermates, that had been administered tamoxifen from postnatal days 1–11. Left: Location of normally flat cells (in control mice) in the superficial zone of the tibial articular cartilage is indicated (black arrows). Right: Note that in Aggrecan1^{tm(IRES-CreERT2)}; Creb5^flox9/flox9 mice, Aggrecan-CreERt2 mediated deletion of exon 9 from Creb5^flox9/flox9 reduces Prg4 expression in the surface of the articular cartilage (yellow arrows) but does not affect Prg4 expression in the synovial membrane (which does not express Aggrecan-CreERt2) (white arrowheads). Femur (F), Tibia (T) and Meniscus (M) are indicated. Scale bar equals 100 microns in a. P21 images were obtained from tissues of female mice. b RNAScope FISH of Col2a1-expressing cells in the knee joint of either a P30 male Aggrecan1^{tm(IRES-CreERT2)}; Creb5^flox9/flox9 mouse or a P30 male Creb5^flox9/flox9 littermate, that had been administered tamoxifen from postnatal days 1–11. Box outlined in left image is enlarged in right image. Arrows indicate small flat Col2a1-expressing cells on the surface of the articular cartilage. Femur (F) and Tibia (T) are indicated. Scale bar equals 200 microns in left panel; 100 microns in right panel. Similar results were obtained in at least 4 (a, b) independent biological repeats. c Quantitation of the number of small flat cells/mm measured surface length of the articular cartilage displayed in b. Each point represents a different RNAScope section obtained from either one P30 male Creb5^flox9/flox9 mouse that was treated with tamoxifen (n = 9 sections); or from one P30 male Aggrecan1^{tm(IRES-CreERT2)}; Creb5^flox9/flox9 mouse that was treated with tamoxifin (n = 8 sections). Statistical analysis was a two-sided test without adjustments. p values are indicated and error bar indicates standard error of the mean. d Creb5 is critical to maintain appropriate expression of both Wnt ligands and other signaling molecules in the interzones of developing synovial joints; and is thus crucial for the formation of most joints. Creb5 both promotes the formation of articular chondrocytes and blocks growth plate development in the epiphyses by inhibiting expression of Wnt5a in the articular/epiphyseal perichondrium.