In the growth plate, stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone and then terminally differentiate into the non-dividing chondrocytes of the hypertrophic zone. To explore the molecular switches responsible for this two-step differentiation program, we developed a microdissection method to isolate RNA from the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ) of 7-day-old male rats. Expression of approximately 29 000 genes was analyzed by microarray and selected genes verified by real-time PCR. The analysis identified genes whose expression changed dramatically during the differentiation program, including multiple genes functionally related to bone morphogenetic proteins (BMPs). BMP-2 and BMP-6 were upregulated in HZ compared with RZ and PZ (30-fold each, P < 0.01 and 0.001 respectively). In contrast, BMP signaling inhibitors were expressed early in the differentiation pathway; BMP-3 and gremlin were differentially expressed in RZ (100- and 80-fold, compared with PZ, P < 0.001 and 0.005 respectively) and growth differentiation factor (GDF)-10 in PZ (160-fold compared with HZ, P < 0.001). Our findings suggest a BMP signaling gradient across the growth plate, which is established by differential expression of multiple BMPs and BMP inhibitors in specific zones. Since BMPs can stimulate both proliferation and hypertrophic differentiation of growth plate chondrocytes, these findings suggest that low levels of BMP signaling in the resting zone may help maintain these cells in a quiescent state. In the lower RZ, greater BMP signaling may help induce differentiation to proliferative chondrocytes. Farther down the growth plate, even greater BMP signaling may help induce hypertrophic differentiation. Thus, BMP signaling gradients may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in growth plate cartilage.

Previous studies of the GH–IGF system gene expression in growth plate using immunohistochemistry and in situ hybridization have yielded conflicting results. We therefore studied the spatial and temporal patterns of mRNA expression of the GH–IGF system in the rat proximal tibial growth plate quantitatively. Growth plates were microdissected into individual zones. RNA was extracted, reverse transcribed and analyzed by real-time PCR. In 1-week-old animals, IGF-I mRNA expression was minimal in growth plate compared with perichondrium, metaphyseal bone, muscle, and liver (70-, 130-, 215-, and 400-fold less). In contrast, IGF-II mRNA was expressed at higher levels than in bone and liver (65- and 2-fold). IGF-II expression was higher in the proliferative and resting zones compared with the hypertrophic zone (P < 0.001). GH receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of IGF-binding proteins (IGFBPs)-1 through -6 mRNA was low throughout the growth plate compared with perichondrium and bone. With increasing age (3-, 6-, 9-, and 12-week castrated rats), IGF-I mRNA levels increased in the proliferative zone (PZ) but remained at least tenfold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically in PZ (780-fold; P < 0.001) whereas, type 2 IGF receptor and IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4 increased significantly with age in growth plate and/or surrounding perichondrium and bone. These data suggest that IGF-I protein in the growth plate is not produced primarily by the chondrocytes themselves. Instead, it derives from surrounding perichondrium and bone. In addition, the decrease in growth velocity that occurs with age may be caused, in part, by decreasing expression of IGF-II and increasing expression of type 2 IGF receptor and multiple IGFBPs.