Genome-wide analyses of gene expression during mouse endochondral ossification

Abstract

Background: Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved.

Methodology/principal findings: To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia.

Conclusions/significance: These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification.

Figure 1. Tibia microdissection and microarray analysis of microdissected zones.

Tibiae from 15.5 day old mouse embryos were dissected into three segments called zones I, II and III. Striated lines indicate location of manual segmentation (A, left panel). Growth plate segments for each zone were pooled and total RNA was isolated and hybridized to Affymetrix MOE 430 2.0 chips containing 45 101 probe sets. This experiment was repeated in quadruplicate (A, right panel). Red lines denote probe sets with increased expression in zone I relative to the baseline signal intensity and blue lines denote probe sets that are decreased relative to the baseline signal intensity. White lines represent genes expressed near the baseline intensity. Growth plate microdissection reveals expected expression profiles for established markers of endochondral bone formation. (B) Microarray expression profiles for chondrocyte differentiation sox family members 5, 6 and 9, (Sox- 5, 6 and 9), collagen 2 (Col2a1), growth differentiation factor 5 (Gdf5), aggrecan 1 (Agc 1), collagen XI alpha 1 (Col11a1), hyaluronan and proteoglycan link protein 1 or cartilage link protein 1(Hapln1), fibroblast growth factor receptor 3 (Fgfr3) and collagen IX alpha 2 (col9a2) are shown (top panel). Late-stage markers of endochondral ossification exhibiting large increases in gene expression in zone III include collagen X (Col10a1), matrix metalloproteinase 13 (Mmp13), matrix -metalloproteinase 9 (Mmp9), tumor necrosis factor (ligand) superfamily, member 11 or receptor activator of NF-kappaB ligand (Tnfsf11), acid phosphatase 5, tartrate resistant (Acp5), integrin binding sialoprotein (Ibsp), dentin matrix protein (Dmp1) and osteopontin (Spp1) (bottom left panel). Expression profiles for later stage markers that exhibit more moderate increases in zone III included colony stimulating factor 1 (Csf1), runt related transcription factor 2 (Runx2), osteomodulin (Omd), a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 4 and 5 (Adamts4, Adamts5) (panel, right panel).

Figure 2. Real-time RT-PCR confirmation of early-stage markers of chondrocyte differentiation.

Expression profiles of known chondrocyte markers were evaluated with qRT-PCR. Zone-specific expression of SRY (sex determining region Y)-box 9 (Sox9), collagen 2 (Col2a1), Indian hedgehog (Ihh) and cyclin-dependent kinase inhibitor 1c (Cdkn1c, encoding p57) profiles are shown on the right, with the corresponding microarray data on the left. Four independent RNA isolations were evaluated for each probe and primer pair and p-values less than 0.05 were deemed significant.

Figure 3. Real-time RT-PCR confirmation of later-stage markers of endochondral ossification.

RNA samples from microdissected embryonic tibiae were used to confirm microarray expression profiles for later-stage chondrocyte markers. Collagen X (Col10a1), bone sialoprotein (Ibsp) and dentin matrix protein 1 (Dmp1) were identified in the microdissected array and validated with qRT-PCR. Four independent RNA isolations were evaluated for each probe and primer pair and p-values less than 0.05 were deemed significant.

Figure 4. Gene ontology annotations of microdissected growth plate zone comparisons.

Lists of probe sets subject to pair-wise comparisons between zones I and zone II (I vs. II), zone II and zone III (II vs. III) and zone I and zone III (I vs. III) were each classified according to biological process (BP), cellular component (CC), and molecular function (MF) (A). The most significant hierarchy was followed in each case until the smallest significant sub-classification was found. In each list, developmental (DEV), collagen (COL) and transporter functions (TRANSF) were identified. P-values less than 0.001 were deemed significant in each case. The number of probe sets both common to each respective zone comparison and unique to a given list was illustrated using a Venn diagram (B).

Figure 5. Genes expressed in both microdissected embryonic tibiae and primary mesenchymal micromass cultures.

Venn diagrams delineating the overlap between probe sets expressed in the microdissected (MD) tibiae array data set and the micromass (MM) array data set (A). Pair-wise comparisons between individual growth plate zones are compared to their similar pair-wise comparisons in the micromass culture data (B). Specifically, probe sets differentially expressed between days 3 and 9 (3 vs. 9) of micromass culture are compared to the I vs. II list. Similarly, probe sets differentially expressed between days 9 and 15 (9 vs. 15) and days 3 and 15 (3 vs. 15) of micromass culture are compared to probes identified in II vs. III and zone I vs. III lists, respectively.

Figure 6. Similarities between the micromass culture time course and microdissected growth plate data sets.

Heat maps of genes exhibiting highest differential expression and positive correlations to either zones I or II in the pair-wise microdissection comparisons are shown. Signal intensities are illustrated by varying shades of red (up-regulation) and blue (down-regulation). Arrows indicate genes common to the corresponding micromass comparisons.

Figure 7. Heat map of micromass culture data set analyzed by GSEA.

GSEA-derived heat maps of the top 100 differentially expressed probe sets enriched in micromass data. Correlations between probe sets and day 3 or day 9 of micromass culture for the first map, day 9 or day 15 for the second map and day 3 or day 15 for the third map are shown. Expression profiles for all experimental replicates are depicted for each time point. Signal intensities are illustrated by varying shades of red (up-regulation) and blue (down-regulation). Arrows indicate genes common to the corresponding microdissected growth plate zone comparisons.