Mouse Growth Plate Cartilage Research Articles

Laser Capture Microdissection of Mouse Growth Plate Cartilage.

The ability to identify, isolate, and study pure populations of cells is critical for understanding normal physiology in organs and tissues, which involves spatial regulation of signaling pathways and interactions between cells with different functions, expression profiles, and lineages. Here, we focus on assessing the growth plate cartilage, composed of multiple functionally and histologically distinct zones, to investigate temporally and spatially dependent gene expression differences. In this chapter, we describe the method of laser capture microdissection to isolate chondrocytes from different zones of differentiation in the mouse growth plate cartilage for RNA isolation, and subsequent downstream applications, such as RNA-sequencing and quantitative real-time PCR. We also provide an assessment of different factors contributing to the integrity of the isolated RNA, such as staining methods and procedures in RNA isolation.

Retinoic acid regulates cell-shape and -death of E-FABP (FABP5)-immunoreactive septoclasts in the growth plate cartilage of mice

Septoclasts, which are mononuclear and spindle-shaped cells with many processes, have been considered to resorb the transverse septa of the growth plate (GP) cartilage at the chondro-osseous junction (COJ). We previously reported the expression of epidermal-type fatty acid-binding protein (E-FABP, FABP5) and localization of peroxisome proliferator-activated receptor (PPAR)β/δ, which mediates the cell survival or proliferation, in septoclasts. On the other hand, retinoic acid (RA) can bind to E-FABP and is stored abundantly in the GP cartilage. From these information, it is possible to hypothesize that RA in the GP is incorporated into septoclasts during the cartilage resorption and regulates the growth and/or death of septoclasts. To clarify the mechanism of the cartilage resorption induced by RA, we administered an overdose of RA or its precursor vitamin A (VA)-deficient diet to young mice. In mice of both RA excess and VA deficiency, septoclasts decreased in the number and cell size in association with shorter and lesser processes than those in normal mice, suggesting a substantial suppression of resorption by septoclasts in the GP cartilage. Lack of PPARβ/δ-expression, TUNEL reaction, RA receptor (RAR)β, and cellular retinoic acid-binding protein (CRABP)-II were induced in E-FABP-positive septoclasts under RA excess, suggesting the growth arrest/cell-death of septoclasts, whereas cartilage-derived retinoic acid-sensitive protein (CD-RAP) inducing the cell growth arrest or morphological changes was induced in septoclasts under VA deficiency. These results support and do not conflict with our hypothesis, suggesting that endogenous RA in the GP is possibly incorporated in septoclasts and utilized to regulate the activity of septoclasts resorbing the GP cartilage.

Expression of epidermal fatty acid binding protein (E-FABP) in septoclasts in the growth plate cartilage of mice.

n-3 Polyunsaturated fatty acids play a role in regulating the growth of the long bones. Fatty acid-binding proteins (FABPs) bind and transport hydrophobic long-chain fatty acids intracellularly, and epidermal-type FABP (E-FABP) has an affinity for n-3 fatty acids. This study aimed to clarify the localization of E-FABP in the growth plate of the mouse tibia. At the chondro-osseous junction (COJ) of the growth plate, E-FABP-immunoreactivity was exclusively localized in mononuclear, spindle-shaped cells with several long processes. These E-FABP-immunoreactive cells were identified as being septoclasts, i.e., cells that resorb uncalcified transverse septa. The processes of these immunoreactive septoclasts terminated between the longitudinal and transverse septa. E-FABP-immunoreactivity was found in the entire cytoplasm and on the mitochondrial outer membrane. In ontogeny, immunoreactive septoclasts were observed immediately after emergence of the primary ossifying center and were distributed not only at the COJ but also in the metaphysis near the COJ. The number of septoclasts increased at the postnatal age of 1 week (P1w)-P2w, and thereafter gradually decreased; and the cells became concentrated at the COJ after P3w-P4w. The immunoreactivity for peroxisome proliferator-activated receptor (PPAR)β/δ was detected in these E-FABP-immunoreactive septoclasts. The present results suggest that fatty acids, preferably n-3 ones, are intracellularly transported by E-FABP to various targets, including mitochondria and nucleus, in which PPARβ/δ may play functional roles in the transcriptional regulation of genes involved in the endochondral ossification.

Predominant expression of H3K9 methyltransferases in prehypertrophic and hypertrophic chondrocytes during mouse growth plate cartilage development

Histone lysine methylation (HKM) is an epigenetic change that establishes cell-specific gene expression and determines cell fates. In this study, we investigated the expression patterns of histone H3 lysine 9 methyltransferases (H3K9MTases) G9a (euchromatic histone lysine N-methyltransferase 2, Ehmt2), GLP (euchromatic histone lysine N-methyltransferase 1, Ehmt1), SETDB1 (SET domain, bifurcated 1), PRDM2 (PR domain containing 2), SUV39H1 (suppressor of variegation 3–9 homolog 1), and SUV39H2, as well as the distribution of 3 types of HKM at histone H3 lysine 9: mono- (H3K9me1), di- (H3K9me2), or tri-methylation (H3K9me3), during mouse growth plate development. In the forelimb cartilage primordial at embryonic day 12.5 (E12.5), none of the H3K9MTases were detected and H3K9me1, H3K9me2, and H3K9me3 were scarcely detected. At E14.5, the H3K9MTases were expressed at low levels in proliferating chondrocytes and at high levels in prehypertrophic and hypertrophic chondrocytes. Among the H3K9 methylations, H3K9me1 and H3K9me3 were markedly noted in these chondrocytes. At E16.5, G9, GLP, SETDB1, PRDM2, SUV39H1, and SUV39H2, as well as H3K9me1, H3K9me2, and H3K9me3, were detected in prehypertrophic and hypertrophic chondrocytes in the growth plate. Western blotting and real-time quantitative polymerase chain reaction analysis revealed the distributions of G9 and GLP proteins and the expression of all the H3K9MTase mRNAs in prehypertrophic and hypertrophic chondrocytes. These data suggest that H3K9 methyltransferases are predominantly expressed in prehypertrophic and hypertrophic chondrocytes, and that they could be involved in the regulation of gene expression and progression of chondrocyte differentiation by affecting the methylation state of histone H3 lysine 9 in the mouse growth plate.

Gene expression profiling of mouse growth plate cartilage by laser microdissection and microarray analysis

Longitudinal bone growth results from a complex sequence of events involving differentiation of resting chondroblasts into proliferative, pre-hypertrophic, and hypertrophic chondrocytes. The growth plate (epiphyseal plate), which is primarily responsible for longitudinal growth, can be divided into four distinct zones: the resting zone (RZ), proliferating zone (PZ), maturing zone (MZ), and hypertrophic zone (HZ), on the basis of the morphology of the developing chondroblasts and the structure of the cartilage matrix. In the past two decades substantial progress has been made in understanding the mechanisms underlying chondroblast differentiation and skeletal development [1–3]. However, comprehensive analysis of gene expression patterns in the growth plate has been technically challenging.

Expression of ADAMTS-9 in mouse growth plate cartilage

Expression of ADAMTS-9 in mouse growth plate cartilage

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.

Proteomic analysis of mouse growth plate cartilage

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.

Twisted Gastrulation Modulates Bone Morphogenetic Protein-induced Collagen II and X Expression in Chondrocytesin Vitroandin Vivo

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.

ADAMTS‐1–Knockout mice do not exhibit abnormalities in aggrecan turnover in vitro or in vivo

To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice. Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses. ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage. Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

Analysis of Osteopontin in Mouse Growth Plate Cartilage by Application of Laser Capture Microdissection and RT-PCR

Gene expression of osteopontin (OPN) has been investigated in mice by application of laser capture microdissection (LCM) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. LCM permits individual cells to be isolated ("captured") from tissue sections for molecular analyses. In this study, chondrocytes were captured from growth plate zones in frozen sections of tibiae from 1-11-day-old postnatal mice. RNA was extracted from cells, DNAse-treated, and reverse-transcribed. cDNA was amplified by PCR and OPN mRNA was revealed on agarose gels. Whole cartilage and brain (a positive control) from the same animals also were examined. Reactions containing no RT were negative controls, and 18S rRNA standardized expressed message from captured cells. RT-PCR analysis of laser-captured whole cartilage showed a general qualitative loss of OPN mRNA as animal age increased. Youngest mice gave equivalent OPN expression over all laser-microdissected cartilage zones. For 7-11 day-old mice, OPN expression was qualitatively greatest in resting and lowest in hypertrophic regions of cartilage. Expression of OPN correlated with mineral in the tissue suggests that OPN functionally may inhibit normal vertebrate growth plate mineralization, and its loss with increasing tissue maturation appears permissive to mineral development.

Analysis of Osteopontin in Mouse Growth Plate Cartilage by Application of Laser Capture Microdissection and RT-PCR

Gene expression of osteopontin (OPN) has been investigated in mice by application of laser capture microdissection (LCM) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. LCM permits individual cells to be isolated ("captured") from tissue sections for molecular analyses. In this study, chondrocytes were captured from growth plate zones in frozen sections of tibiae from 1-11-day-old postnatal mice. RNA was extracted from cells, DNAse-treated, and reverse-transcribed. cDNA was amplified by PCR and OPN mRNA was revealed on agarose gels. Whole cartilage and brain (a positive control) from the same animals also were examined. Reactions containing no RT were negative controls, and 18S rRNA standardized expressed message from captured cells. RT-PCR analysis of laser-captured whole cartilage showed a general qualitative loss of OPN mRNA as animal age increased. Youngest mice gave equivalent OPN expression over all laser-microdissected cartilage zones. For 7-11 day-old mice, OPN expression was qualitatively greatest in resting and lowest in hypertrophic regions of cartilage. Expression of OPN correlated with mineral in the tissue suggests that OPN functionally may inhibit normal vertebrate growth plate mineralization, and its loss with increasing tissue maturation appears permissive to mineral development.

Expression and Signal Transduction of Calcium-Sensing Receptors in Cartilage and Bone1

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.