Indian Hedgehog Signaling in the Postnatal Growth Plate
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ABSTRACT: In order to characterize mRNA expression in the growth plate, we microdissected postnatal rat growth plates into their constituent zones and used microarray analysis to assess the abundences of individual transcripts. Expression patterns of PTHrP and Ihh-related genes were confirmed using real-time PCR. Using a gli1-lacZ mouse, Gli1 expression, presumably representing Ihh signaling, was visualized during pre- and postnatal development. Microdissection was used to collect individual growth plate zones from proximal tibiae of 1-wk rats and gene expression was analyzed using microarray.
Project description:To explore the mechanisms responsible for spatial and temporal regulation of the growth plate, we microdissected postnatal rat growth plates into their constituent zones and then used microarray analysis to characterize the changes in gene expression that occur as chondrocytes undergo spatially-associated differentiation and temporally-associated senescence. Microdissection was used to collect individual growth plate zones from proximal tibiae of 1-wk rats and the proliferative and early hypertrophic zones of growth plates from 3-, 6-, 9-, and 12-wk rats and gene expression was analyzed using microarray.
Project description:Comparison of human prepuberal articular and growth plate cartilage Total RNA was isolated from healthy human prepuberal tibiae and femurs donors
Project description:In order to characterize mRNA expression in the growth plate, we microdissected postnatal rat growth plates into their constituent zones and used microarray analysis to assess the abundences of individual transcripts. Expression patterns of PTHrP and Ihh-related genes were confirmed using real-time PCR. Using a gli1-lacZ mouse, Gli1 expression, presumably representing Ihh signaling, was visualized during pre- and postnatal development.
Project description:In progressed puberty, estrogen is responsible for the deceleration of growth by stimulating growth plate maturation. The mechanism of action is largely unknown. We obtained pubertal growth plate specimens of the same girl at Tanner stage B2 and Tanner stage B3, which allowed us to address this issue in more detail. Histological analysis showed that progression of puberty coincided with characteristic morphological changes associated with growth plate maturation, such as decreases in total growth plate height (p=0.002), height of the individual zones (p<0.001) and a increase in intercolumnar space (p<0.001). Microarray analysis of the specimens identified 394 genes (72% upregulated, 28% downregulated) changing with progression of puberty. Overall changes in gene expression were small (average 1.1 fold change). The 394 genes mapped to 13 significantly changing pathways (p<0.05) in majority belonging to extracellular matrix, cell cycle and cell death, which are all related to growth plate maturation. We next scanned the upstream promoter regions of the 394 genes for the presence of evolutionary conserved binding sites for transcription factors implemented in growth plate maturation such as Estrogen Receptor, Androgen Receptor, Elk1, Stat5b, CREBP and Runx2. Runx2 and Elk1, but not estrogen receptor binding sites were enriched and were present in 87 and 43 out of the 394 genes, respectively.In conclusion, our data suggest a role for Runx2 and Elk1 in growth plate maturation and provides suggestive evidence that the effect of estrogen on growth plate maturation is not mediated by activating genomic estrogen signalling in growth plate chondrocytes. 2 replicate samples for each developmental stage for a total of 6 samples
Project description:Previous studies demonstrated that hererozygous missense mutations in IHH could lead to a decreasing IHH signaling efficacy and cause brachydactyly type A1 (BDA1). For revealing difference of Gli1-mediated downstream regulatory effects between wild type IHH signaling and E95K mutant IHH signaling, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to be targets of Gli1 cis-regulation.
Project description:Previous studies demonstrated that hererozygous missense mutations in IHH could lead to a decreasing IHH signaling efficacy and cause brachydactyly type A1 (BDA1). For revealing difference of Gli1-mediated downstream regulatory effects between wild type IHH signaling and E95K mutant IHH signaling, we have employed whole genome microarray miRNA expression profiling as a discovery platform to identify miRNAs with the potential to take part in Gli1-mediated cis-regulatory network.
Project description:Indian Hedgehog signaling (IHH) regulates multiple aspects of endochondral bone formation and ultimately regulates expression of downstream genes by Gli zinc finger domain-containing transcription regulators in the cartilage development. In response to a BDA1 mutant IHH induction, Gli1-mediated downstream regulatory pattern might be quite different from that in response to wild type IHH induction.
Project description:Suture stem cells (SuSCs) are important for homeostasis and regeneration of cranial bone and can be used as a model to understand stem cell regulation for bone regeneration at a distance. Using a mouse cranial bone injury model, we identified the chemokine Cxcl12 and the Hedgehog family ligands Shh and Ihh as injury-induced interacting niche factors that coordinately regulate Gli1+ SuSC expansion, directional migration,and osteoblastic differentiation. Cxcl12 increased in the early inflammatory phase after injury, and Cxcl12-mediated activation of its cognate receptor, Cxcr4, in Gli1+ SuSCs promoted bone regeneration by stimulating SuSC proliferation and migration. Cxcl12-Cxcr4 signaling induced Shh and Ihh expression, which promoted Gli1+ SuSC proliferation and osteoblastic differentiation.
Project description:Articular cartilage is deprived of blood vessels and nerves, and the only cells residing in this tissue are chondrocytes. The molecular properties of the articular cartilage and the architecture of the extracellular matrix demonstrate a complex structure that differentiates on the depth of tissue. Osteoarthritis (OA) is a degenerative joint disease, the most common form of arthritis, affecting the whole joint. It is associated with ageing and affects the joints that have been continually stressed throughout life including the knees, hips, fingers, and lower spine region. OA is a multifactorial condition of joint characterised by articular cartilage loss, subchondral bone sclerosis, and inflammation leading to progressive joint degradation, structural alterations, loss of mobility and pain. Articular cartilage biology is well studied with a focus on musculoskeletal diseases and cartilage development. However, there are relatively few studies focusing on zonal changes in the cartilage during osteoarthritis.
Project description:Objective. Identify novel genes and pathways specific to superficial (SZ), middle (MZ) and deep zones (DZ) of normal articular cartilage. Methods. Articular cartilage was obtained from knees of 4 normal human donors. The cartilage zones were dissected on a microtome. RNA was analyzed on human genome arrays. Data obtained with human tissue were compared to bovine cartilage zone specific DNA arrays. Genes differentially expressed between zones were evaluated using direct annotation for structural or functional features, and by enrichment analysis for integrated pathways or functions. Results. The greatest differences were observed between SZ and DZ in both human and bovine cartilage. The MZ was transitional between the SZ and DZ and thereby shared some of the same pathways as well as structural/functional features of the adjacent zones. Cellular functions and biological processes enriched in the SZ relative to the DZ, include most prominently ECM receptor interactions, cell adhesion molecules, regulation of actin cytoskeleton, ribosome-related functions and signaling aspects such as Interferon gamma, IL4, CDC42Rac and Jak-Stat. Two pathways were enriched in the DZ relative to the SZ, including PPARG and EGFR_SMRTE. Conclusion. These differences in cartilage zonal gene expression identify new markers and pathways that govern the unique differentiation status of chondrocyte subpopulations. 6 samples, 2 bovine donors, 3 conditions each donor (SZ, MZ and DZ), 0 donor replicates, comparisons made between SZ, MZ and DZ to identify differentially expressed genes.