Dataset Information

Spatial regulation of gene expression during growth of articular cartilage in juvenile mice.

ABSTRACT: BACKGROUND:In juvenile mammals, the epiphyses of long bones grow by chondrogenesis within the articular cartilage. A better understanding of the molecular mechanisms that regulate the growth of articular cartilage may give insight into the antecedents of joint disease, such as osteoarthritis. METHODS:We used laser capture microdissection to isolate chondrocytes from the superficial, middle, and deep zones of growing tibial articular cartilage in the 1-wk-old mouse and then investigated expression patterns by microarray. To identify molecular markers for each zone of the growing articular cartilage, we found genes showing zone-specific expression and confirmed by real-time PCR and in situ hybridization. RESULTS:Bioinformatic analyses implicated ephrin receptor signaling, Wnt signaling, and bone morphogenetic protein signaling in the spatial regulation of chondrocyte differentiation during growth. Molecular markers were identified for superficial (e.g., Cilp, Prg4), middle (Cxcl14, Tnn), and deep zones (Sfrp5, Frzb). Comparison between juvenile articular and growth plate cartilage revealed that the superficial-to-deep zone transition showed similarity with the hypertrophic-to-resting zone transition. CONCLUSION:Laser capture microdissection combined with microarray analysis identified novel signaling pathways that are spatially regulated in growing mouse articular cartilage and revealed similarities between the molecular architecture of the growing articular cartilage and that of growth plate cartilage.

SUBMITTER: Lui JC

PROVIDER: S-EPMC6354579 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Spatial regulation of gene expression during growth of articular cartilage in juvenile mice.

Lui Julian C JC Chau Michael M Chen Weiping W Cheung Crystal S F CS Hanson Jeffrey J Rodriguez-Canales Jaime J Nilsson Ola O Baron Jeffrey J

Pediatric research 20141218 3

<h4>Background</h4>In juvenile mammals, the epiphyses of long bones grow by chondrogenesis within the articular cartilage. A better understanding of the molecular mechanisms that regulate the growth of articular cartilage may give insight into the antecedents of joint disease, such as osteoarthritis.<h4>Methods</h4>We used laser capture microdissection to isolate chondrocytes from the superficial, middle, and deep zones of growing tibial articular cartilage in the 1-wk-old mouse and then investi ...[more]

PMID: 25521919

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Project description:Articular and growth plate cartilage are discrete tissues but arise from a common cartilaginous condensation and have comparable spatial architectures consisting of distinct layers of chondrocytes. To investigate similarities and differences between articular and growth plate cartilage and to explore transcriptional changes that occur during the onset of their divergence, we performed manual microdissection of 10-day-old rat proximal tibias, microarray analysis, bioinformatics, and real-time PCR to compare gene expression profiles in individual cartilage layers. We found that many genes that were spatially upregulated in the intermediate/deep zone of articular cartilage were also spatially upregulated in the resting zone of growth plate cartilage (overlap greater than expected by chance, P<0.001). Interestingly, the superficial zone of articular cartilage showed an expression profile with similarities to both the proliferative and hypertrophic zones of growth plate cartilage (P<0.001 each). Additionally, significant numbers of known proliferative zone markers (3 out of 6) and hypertrophic zone markers (27 out of 126) were spatially upregulated in the superficial zone (more than expected by chance, P<0.001 each). In conclusion, we provide evidence that the intermediate/deep zone of articular cartilage has a gene expression profile more similar to that of the resting zone of growth plate cartilage, whereas the superficial zone has a gene expression profile more similar to those of the proliferative and hypertrophic zones. These findings suggest that the superficial zone chondrocytes of articular cartilage differentiate according to a program that is not completely different from but instead has distinct similarities to the hypertrophic differentiation program of growth plate chondrocytes. We also present functional signaling pathways implicated by differential gene expression between articular and growth plate cartilage during their initial separation by the secondary ossification center.

Project description:Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers-the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.

Project description:BackgroundArticular cartilage undergoes an important maturation process from neonate to adult that is reflected by alterations in matrix protein organization and increased heterogeneity of chondrocyte morphology. In the horse, these changes are influenced by exercise during the first five months of postnatal life. Transcriptional profiling was used to evaluate changes in articular chondrocyte gene expression during postnatal growth and development.MethodsTotal RNA was isolated from the articular cartilage of neonatal (0-10 days) and adult (4-5 years) horses, subjected to one round of linear RNA amplification, and then applied to a 9,367-element equine-specific cDNA microarray. Comparisons were made with a dye-swap experimental design. Microarray results for selected genes (COL2A1, COMP, P4HA1, TGFB1, TGFBR3, TNC) were validated by quantitative polymerase chain reaction (qPCR).ResultsFifty-six probe sets, which represent 45 gene products, were up-regulated (p < 0.01) in chondrocytes of neonatal articular cartilage relative to chondrocytes of adult articular cartilage. Conversely, 586 probe sets, which represent 499 gene products, were up-regulated (p < 0.01) in chondrocytes of adult articular cartilage relative to chondrocytes of neonatal articular cartilage. Collagens, matrix-modifying enzymes, and provisional matrix non-collagenous proteins were expressed at higher levels in the articular cartilage of newborn foals. Those genes with increased mRNA abundance in adult chondrocytes included leucine-rich small proteoglycans, matrix assembly, and cartilage maintenance proteins.ConclusionDifferential expression of genes encoding matrix proteins and matrix-modifying enzymes between neonates and adults reflect a cellular maturation process in articular chondrocytes. Up-regulated transcripts in neonatal cartilage are consistent with growth and expansion of the articular surface. Expression patterns in mature articular cartilage indicate a transition from growth to homeostasis, and tissue function related to withstanding shear and weight-bearing stresses.

Dataset Information

Spatial regulation of gene expression during growth of articular cartilage in juvenile mice.

Publications

Spatial regulation of gene expression during growth of articular cartilage in juvenile mice.

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