Modeling human skeletal development using human pluripotent stem cells.

Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice.

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER-associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3-week-old male OI Col1a2 +/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 +/p.G610C mouse bone structure, strength or composition, measured by micro-computed tomography, three point bending tests and Fourier-transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.

OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

CRISPR/Cas9 gene editing of a SOX9 reporter human iPSC line to produce two TRPV4 patient heterozygous missense mutant iPSC lines, MCRIi001-A-3 (TRPV4 p.F273L) and MCRIi001-A-4 (TRPV4 p.P799L).

To produce in vitro models of human chondrodysplasias caused by dominant missense mutations in TRPV4, we used CRISPR/Cas9 gene editing to introduce two heterozygous patient mutations (p.F273L and p.P799L) into an established control human iPSC line. This control line expressed a fluorescent reporter (tdTomato) at the SOX9 locus to allow real-time monitoring of cartilage differentiation by SOX9 expression. Both TRPV4 mutant iPSC lines had normal karyotypes, expressed pluripotency markers, and could differentiate into cells representative of the three embryonic germ layers. These iPSC lines, with the parental isogenic control, will be used to study TRPV4 chondrodysplasia mechanisms and explore therapeutic approaches.

Cartilage MicroRNA Dysregulation During the Onset and Progression of Mouse Osteoarthritis Is Independent of Aggrecanolysis and Overlaps With Candidates From End-Stage Human Disease.

OBJECTIVE: To identify candidate microRNAs (miRNAs) that potentially regulate the initiation and progression of osteoarthritis (OA). METHODS: OA was induced in 10-12-week-old male wild-type C57BL/6 mice and in mice resistant to aggrecanase cleavage (Acan p.374ALGS→374NVYS) by destabilization of the medial meniscus (DMM). Pathologic changes of OA were scored histologically. RNA from cartilage and subchondral bone was harvested in parallel by laser microdissection at 1 week and 6 weeks postsurgery. Global miRNA expression profiling was performed using Agilent microarrays and was validated by quantitative polymerase chain reaction analysis. RESULTS: Wild-type DMM mice had characteristic cartilage degeneration, subchondral bone sclerosis, and osteophyte formation. While no miRNA dysregulation was seen in subchondral bone, 139 miRNAs were differentially expressed in cartilage obtained at 1 and/or 6 weeks after OA initiation from wild-type mice that underwent DMM. To prioritize OA candidates, dysregulated miRNAs with human orthologs were filtered, and paired miRNA/messenger RNA (mRNA) expression analysis was conducted to identify those with corresponding changes in mRNA target transcripts in the DMM mouse cartilage. An important cohort also overlapped with miRNAs identified in human end-stage OA. Comparisons of miRNA dysregulation in DMM mouse cartilage where aggrecan cleavage was genetically ablated demonstrated that all candidates were independent of aggrecan breakdown, earmarking these as important to the critical stages of OA initiation. Furthermore, functional enrichment analysis and data annotation revealed the responses to mechanical stimuli, apoptotic processes, and core extracellular matrix structural and regulatory factors to be potentially influenced by OA-dysregulated miRNA/mRNA networks. CONCLUSION: Our comprehensive analyses identified high-priority miRNA candidates that have potential as biomarkers and therapeutic targets in human OA.

Comprehensive Expression Analysis of microRNAs and mRNAs in Synovial Tissue from a Mouse Model of Early Post-Traumatic Osteoarthritis.

To better understand the molecular processes involved in driving osteoarthritis disease progression we characterized expression profiles of microRNAs (miRNA) and mRNAs in synovial tissue from a post-traumatic OA mouse model. OA was induced in 10-12 week old male C57BL6 mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA isolated from the anterior synovium of mice at 1 and 6 weeks post-surgery was subject to expression profiling using Agilent microarrays and qPCR. OA severity was determined histologically. Anterior and posterior synovitis decreased with post-operative time after sham and DMM. No differences in synovitis parameters were evident between sham and DMM in the anterior synovium at either time. While expression profiling revealed 394 miRNAs were dysregulated between 1 and 6 week time-points in the anterior synovium, there were no significant changes in miRNA or mRNA expression between DMM and sham mice at both time-points. Bioinformatic analysis of the miRNAs and mRNAs differentially expressed in tandem with the resolution of anterior synovial inflammation revealed similar biological processes and functions, including organismal injury, connective tissue disorder and inflammatory responses. Our data demonstrates that early OA-specific patterns of synovial miRNAs or mRNAs dysregulation could not be identified in this model of post-traumatic OA.

Novel Elements of the Chondrocyte Stress Response Identified Using an in Vitro Model of Mouse Cartilage Degradation.

The destruction of articular cartilage in osteoarthritis involves chondrocyte dysfunction and imbalanced extracellular matrix (ECM) homeostasis. Pro-inflammatory cytokines such as interleukin-1α (IL-1α) contribute to osteoarthritis pathophysiology, but the effects of IL-1α on chondrocytes within their tissue microenvironment have not been fully evaluated. To redress this we used label-free quantitative proteomics to analyze the chondrocyte response to IL-1α within a native cartilage ECM. Mouse femoral heads were cultured with and without IL-1α, and both the tissue proteome and proteins released into the media were analyzed. New elements of the chondrocyte response to IL-1α related to cellular stress included markers for protein misfolding (Armet, Creld2, and Hyou1), enzymes involved in glutathione biosynthesis and regeneration (Gstp1, Gsto1, and Gsr), and oxidative stress proteins (Prdx2, Txn, Atox1, Hmox1, and Vnn1). Other proteins previously not associated with the IL-1α response in cartilage included ECM components (Smoc2, Kera, and Crispld1) and cysteine proteases (cathepsin Z and legumain), while chondroadherin and cartilage-derived C-type lectin (Clec3a) were identified as novel products of IL-1α-induced cartilage degradation. This first proteome-level view of the cartilage IL-1α response identified candidate biomarkers of cartilage destruction and novel targets for therapeutic intervention in osteoarthritis.

The circadian clock in murine chondrocytes regulates genes controlling key aspects of cartilage homeostasis.

OBJECTIVE: To characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA). METHODS: Cartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription-polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis. RESULTS: Mouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA. CONCLUSION: These results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease.

Mechanistic insight into the pathology of polyalanine expansion disorders revealed by a mouse model for X linked hypopituitarism.

Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.

Maintaining mRNA integrity during decalcification of mineralized tissues.

Biomineralization of the extracellular matrix occurs inappropriately in numerous pathological conditions such as cancer and vascular disease, but during normal mammalian development calcification is restricted to the formation of the skeleton and dentition. The comprehensive study of gene expression in mineralized skeletal tissues has been compromized by the traditional decalcification/fixation methods that result in significant mRNA degradation. In this study we developed a novel RNAlater/EDTA decalcification method that protects the integrity of the mRNA in mature mouse tibial epiphyses. Furthermore, this method preserves the tissue structure to allow histological sectioning and microdissection to determine region-specific gene expression, in addition to immuno- and in situ histology. This method will be widely applicable to the molecular analysis of calcified tissues in various pathological conditions, and will be of particular importance in dissection of the gene expression in mouse bone and joint tissues during development and in important clinical conditions such as arthritis.

Transcriptomics of wild-type mice and mice lacking ADAMTS-5 activity identifies genes involved in osteoarthritis initiation and cartilage destruction.

OBJECTIVE: To identify changes in gene expression in mice with osteoarthritis (OA) in order to explore the mechanisms of the disease. METHODS: Gene expression profiling was performed in cartilage from mice with surgically induced OA. We used wild-type (WT) mice and Adamts5Δcat mice, in which ADAMTS-5 activity is lacking and aggrecan loss and cartilage erosion are inhibited, to distinguish gene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown. Mechanical instability was introduced into the knee joints of 10-week-old male mice via surgical destabilization of the medial meniscus (DMM). Cartilage from the developing lesion in the destabilized medial meniscus and corresponding regions in sham-operated joints was harvested by microdissection at 1, 2, and 6 weeks postsurgery, and RNA was extracted, amplified, and hybridized to whole-genome microarrays. RESULTS: Several previously identified OA-related genes, including Ptgs2, Crlf1, and Inhba, and novel genes, such as Phdla2 and Il11, were up-regulated in both WT mice and Adamts5Δcat mice, indicating that they are independent of ADAMTS-5 activity. The altered expression of other genes, including Col10a1, the sentinel marker of cartilage hypertrophy, and Wnt/ß-catenin pathway genes, required ADAMTS-5 activity. Cell death pathway genes were dysregulated, and Tp53, Foxo4, and Xbp1 endoplasmic reticulum-stress transcriptional networks were activated. Analysis of degradome genes identified up-regulation of many proteases, including Mmp3, Capn2, and the novel cartilage proteases Prss46 and Klk8. Comparison with other studies identified 16 genes also dysregulated in rat and human OA as priorities for study. CONCLUSION: We have identified, for the first time, several genes that have an ADAMTS-5-independent role in OA, identifying them as possible OA initiation candidates. This work provides new insights into the sequence of gene dysregulation and the molecular basis of cartilage destruction in OA.

Mutations in TRPV4 cause an inherited arthropathy of hands and feet.

Familial digital arthropathy-brachydactyly (FDAB) is a dominantly inherited condition that is characterized by aggressive osteoarthropathy of the fingers and toes and consequent shortening of the middle and distal phalanges. Here we show in three unrelated families that FDAB is caused by mutations encoding p.Gly270Val, p.Arg271Pro and p.Phe273Leu substitutions in the intracellular ankyrin-repeat domain of the cation channel TRPV4. Functional testing of mutant TRPV4 in HEK-293 cells showed that the mutant proteins have poor cell-surface localization. Calcium influx in response to the synthetic TRPV4 agonists GSK1016790A and 4αPDD was significantly reduced, and mutant channels did not respond to hypotonic stress. Others have shown that gain-of-function TRPV4 mutations cause skeletal dysplasias and peripheral neuropathies. Our data indicate that TRPV4 mutations that reduce channel activity cause a third phenotype, inherited osteoarthropathy, and show the importance of TRPV4 activity in articular cartilage homeostasis. Our data raise the possibility that TRPV4 may also have a role in age- or injury-related osteoarthritis.

Cartilage intermediate layer protein 2 (CILP-2) is expressed in articular and meniscal cartilage and down-regulated in experimental osteoarthritis.

Using transcriptome profiling to determine differential gene expression between the permanent mouse articular cartilage and the transient growth plate cartilage, we identified a highly expressed gene, Cilp2, which is expressed differentially by articular chondrocytes. CILP-2 is highly homologous to CILP-1 (cartilage intermediate layer protein 1), which is expressed in the intermediate zone of articular cartilage and has been linked to cartilage degenerative diseases. We demonstrated that Cilp2 has a restricted mRNA distribution at the surface of the mouse articular cartilage during development, becoming localized to the intermediate zone of articular cartilage and meniscal cartilage with maturity. Although the extracellular CILP-2 protein localization is broadly similar to CILP-1, CILP-2 appears to be more localized in the deeper intermediate zone of the articular cartilage extracellular matrix at maturity. CILP-2 was shown to be proteolytically processed, N-glycosylated, and present in human articular cartilage. In surgically induced osteoarthritis in mice, Cilp1 and Cilp2 gene expression was dysregulated. However, whereas Cilp1 expression was increased, Cilp2 gene expression was down-regulated demonstrating a differential response to mechanically induced joint destabilization. CILP-2 protein was reduced in the mouse osteoarthritic cartilage. Ultrastructural analysis also suggested that CILP-2 may be associated with collagen VI microfibrils and thus may mediate interactions between matrix components in the territorial and inter-territorial articular cartilage matrix. mRNA expression analysis indicated that whereas Cilp1 and Cilp2 are expressed most abundantly in cartilaginous tissues, expression can be detected in muscle and heart.

Identification of SOX3 as an XX male sex reversal gene in mice and humans.

Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome-linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box-containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.

S100A8 and S100A9 in experimental osteoarthritis.

INTRODUCTION: The objective was to evaluate the changes in S100A8 S100A9, and their complex (S100A8/S100A9) in cartilage during the onset of osteoarthritis (OA) as opposed to inflammatory arthritis. METHODS: S100A8 and S100A9 protein localization were determined in antigen-induced inflammatory arthritis in mice, mouse femoral head cartilage explants stimulated with interleukin-1 (IL-1), and in surgically-induced OA in mice. Microarray expression profiling of all S100 proteins in cartilage was evaluated at different times after initiation of degradation in femoral head explant cultures stimulated with IL-1 and surgically-induced OA. The effect of S100A8, S100A9 or the complex on the expression of aggrecan (Acan), collagen II (Col2a1), disintegrin and metalloproteases with thrombospondin motifs (Adamts1, Adamts 4 &Adamts 5), matrix metalloproteases (Mmp1, Mmp3, Mmp13 &Mmp14) and tissue inhibitors of metalloproteinases (Timp1, Timp2 &Timp3), by primary adult ovine articular chondrocytes was determined using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Stimulation with IL-1 increased chondrocyte S100a8 and S100a9 mRNA and protein levels. There was increased chondrocyte mRNA expression of S100a8 and S100a9 in early but not late mouse OA. However, loss of the S100A8 staining in chondrocytes occurred as mouse OA progressed, in contrast to the positive reactivity for both S100A8 and S100A9 in chondrocytes in inflammatory arthritis in mice. Homodimeric S100A8 and S100A9, but not the heterodimeric complex, significantly upregulated chondrocyte Adamts1, Adamts4 and Adamts 5, Mmp1, Mmp3 and Mmp13 gene expression, while collagen II and aggrecan mRNAs were significantly decreased. CONCLUSIONS: Chondrocyte derived S100A8 and S100A9 may have a sustained role in cartilage degradation in inflammatory arthritis. In contrast, while these proteins may have a role in initiating early cartilage degradation in OA by upregulating MMPs and aggrecanases, their reduced expression in late stages of OA suggests they do not have an ongoing role in cartilage degradation in this non-inflammatory arthropathy.

Mutations in the heparan-sulfate proteoglycan glypican 6 (GPC6) impair endochondral ossification and cause recessive omodysplasia.

Glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored, membrane-bound heparan sulfate (HS) proteoglycans. Their biological roles are only partly understood, although it is assumed that they modulate the activity of HS-binding growth factors. The involvement of glypicans in developmental morphogenesis and growth regulation has been highlighted by Drosophila mutants and by a human overgrowth syndrome with multiple malformations caused by glypican 3 mutations (Simpson-Golabi-Behmel syndrome). We now report that autosomal-recessive omodysplasia, a genetic condition characterized by short-limbed short stature, craniofacial dysmorphism, and variable developmental delay, maps to chromosome 13 (13q31.1-q32.2) and is caused by point mutations or by larger genomic rearrangements in glypican 6 (GPC6). All mutations cause truncation of the GPC6 protein and abolish both the HS-binding site and the GPI-bearing membrane-associated domain, and thus loss of function is predicted. Expression studies in microdissected mouse growth plate revealed expression of Gpc6 in proliferative chondrocytes. Thus, GPC6 seems to have a previously unsuspected role in endochondral ossification and skeletal growth, and its functional abrogation results in a short-limb phenotype.

Mice lacking the extracellular matrix protein WARP develop normally but have compromised peripheral nerve structure and function.

WARP is a recently identified extracellular matrix molecule with restricted expression in permanent cartilages and a distinct subset of basement membranes in peripheral nerves, muscle, and the central nervous system vasculature. WARP interacts with perlecan, and we also demonstrate here that WARP binds type VI collagen, suggesting a function in bridging connective tissue structures. To understand the in vivo function of WARP, we generated a WARP-deficient mouse strain. WARP-null mice were healthy, viable, and fertile with no overt abnormalities. Motor function and behavioral testing demonstrated that WARP-null mice exhibited a significantly delayed response to acute painful stimulus and impaired fine motor coordination, although general motor function was not affected, suggesting compromised peripheral nerve function. Immunostaining of WARP-interacting ligands demonstrated that the collagen VI microfibrillar matrix was severely reduced and mislocalized in peripheral nerves of WARP-null mice. Further ultrastructural analysis revealed reduced fibrillar collagen deposition within the peripheral nerve extracellular matrix and abnormal partial fusing of adjacent Schwann cell basement membranes, suggesting an important function for WARP in stabilizing the association of the collagenous interstitial matrix with the Schwann cell basement membrane. In contrast, other WARP-deficient tissues such as articular cartilage, intervertebral discs, and skeletal muscle showed no detectable abnormalities, and basement membranes formed normally. Our data demonstrate that although WARP is not essential for basement membrane formation or musculoskeletal development, it has critical roles in the structure and function of peripheral nerves.

A microarray approach for comparative expression profiling of the discrete maturation zones of mouse growth plate cartilage.

In vertebrates, longitudinal bone growth is the consequence of a complex series of events that take place in a specialized structure, the growth plate cartilage. Within the growth plate chondrocytes undergo a sequential maturation program from resting cells to proliferative, pre-hypertrophic, and ultimately hypertrophic end-stage chondrocytes. This process of chondrocyte maturation is under the control of the temporally and spatially regulated expression of a myriad of signaling molecules, transmembrane receptors, transcription factors, and structural extracellular matrix (ECM) proteins. One approach to the comprehensive definition of the key components of such complex interrelated pathways is the use of microarray expression profiling to catalogue transcriptome changes during chondrocyte maturation in the individual developmental zones of the mouse growth plate cartilage. However, this has not been achieved because of the difficulty in obtaining sufficient quantities of the individual growth plate cartilage zones to all microarray analysis. In this study we describe the development of microdissection methods for the isolation of tissue from the proliferative, pre-hypertrophic, and proliferative zone from one single mouse femur, RNA extraction and linear amplification of the RNA to allow interrogation of NIA 15k microarrays to generate comparative expression profiles. Verification of a subset of differentially expressed genes by RT-PCR and by in situ hybridization confirmed the reliability of this approach.