Differences in specific paracrine signaling pathways contribute to disparities in growth plate function between different bones.

<p>RNA-Seq was used to identify genes differentially expressed between proximal tibia and proximal phalanx (>2-fold, FDR < 0.05, both species, age 1 week). (A) Gene ontology and signaling pathway analyses showed enrichment for developmental-related functions and identified pathways previously implicated in growth plate biology. (B, C) Heatmaps were generated by hierarchical clustering of the 150 genes that showed the greatest differential expression between tibia and phalanx. Scale bar represents log₂ (fold differences). (D–F) Specific genes from IGF (panel D), BMP (panel E), and Wnt (panel F) signaling pathways that showed significant differential expression between 1-week tibia and phalanx by RNA-Seq were selected for validation by qPCR and to determine time course. (G, H) When cultured in monolayer, primary mouse chondrocytes isolated from tibias showed higher Igf2 expression (panel G, light green bars) and more rapid proliferation (panel H, open light green bars) than those from phalanges. Proliferation of phalangeal chondrocytes, but not tibial chondrocytes, responded positively to exogenous Igf1 (panel H, striped light green bars). When treated with siRNA against Igf2 (no exogenous Igf1; panel G, dark green bars), proliferation was inhibited (versus control) in tibial chondrocytes but not phalangeal chondrocytes (panel H, open dark green bars). This inhibition was partially reversed by exogenous Igf1 (panel H, striped dark green bar). (I) Western blot showed higher levels of p-SMAD1/5/9 in tibial/femoral growth plate chondrocytes than metacarpal/phalangeal chondrocytes, implying more active BMP signaling in the longer bones. (J, K) Neonatal mouse tibias and metatarsals were treated with Bmp2 or Noggin in culture for 3 days, followed by histological examination for chondrocyte hypertrophy. Scale bar, 50 μm. <i>N</i> = 8–10; horizontal line represents sample means. Raw values for Fig 4D–4H, and K are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005263#pbio.2005263.s020" target="_blank">S1 Data</a>. BMP, bone morphogenetic protein; FDR, false discovery rate; IGF, insulin-like growth factor; pSMAD1/5/9, phosphorylated SMAD1/5/9; qPCR, quantitative PCR; RNA-Seq, RNA sequencing; siRNA, small interfering RNA; Wnt, Wingless and Int-1.