Transcription profiling of mouse growth plate chondrocyte differentiation yields insight into endochondral ossification

A variety of cell cultures models and in vivo approaches have been used to study gene expression during chondrocyte differentiation. The extent to which the in vitro models reflect bona fide gene regulation in the growth plate has not been quantified. In addition, studies that evaluate global gene expression changes among different growth plate zones are limited. To address these issues, we completed a microarray screen of three growth plate zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective growth plate zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs confirmed documented data and pointed to novel aspects of chondrocyte differentiation. Parallel comparisons of the microdissected tibiae data set to 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 growth plate. These studies will allow us to better understand zone-specific gene expression patterns in the growth plate. Ultimately, this work will help define both the genomic context in which genes are expressed in the long bones and the extent to which the micromass culture system is able to recapitulate chondrocyte development in endochondral ossification. Experiment Overall Design: Tibiae from 15.5 day old mouse embryos were isolated and partitioned into three distinct zones. Total RNA was isolated from each segment and the individual segments pooled within a litter. Experiment Overall Design: Samples were hybridized to Affymetrix MOE 430 2.0 mouse chips for analysis. Four independent trials were executed for each zone. Experiment Overall Design: Number of time points: 1 Experiment Overall Design: Number of treatments: 0 Experiment Overall Design: Number of Samples: 4 replicates per zone Experiment Overall Design: Affymetrix chip: MOE 430 2.0 Experiment Overall Design: Tissue or origin: Tibiae Experiment Overall Design: Species E15.5 mice Experiment Overall Design: Samples: Total RNA为研究软骨细胞分化（chondrocyte differentiation）过程中的基因表达调控，学界已采用多种体外细胞培养模型与体内实验方法。然而，体外模型在多大程度上能够真实反映生长板（growth plate）内的基因调控状态，目前尚未被量化；同时，针对不同生长板区域间全局基因表达变化的研究也较为有限。为解决上述问题，本研究对手动分割的胚胎小鼠胫骨的三个生长板区域开展了微阵列（microarray）筛查。通过对各区域间差异表达基因进行分类、功能注释，并表征其基因表达模式、细胞遗传学位点（cytogenetic loci）、信号通路（signaling pathways）及功能基序（functional motifs），本研究不仅验证了已有研究数据，还揭示了软骨细胞分化的全新调控维度。将显微切割胫骨的数据集与本团队此前完成的微团培养（micromass culture）筛查结果进行平行比对，进一步证实了微团培养模型用于模拟软骨细胞发育过程中基因表达的适用性。尽管微团培养体系与体内显微切割胫骨筛查结果呈现出高度相似性，但该体系无法精准区分生长板肥大区（hypertrophic zone）与矿化区（mineralized zone）的基因表达差异。本研究有助于更深入地理解生长板区域特异性的基因表达模式。最终，本研究将明确长骨中基因表达的基因组背景，以及微团培养体系在软骨内骨化（endochondral ossification）过程中重现软骨细胞发育的能力范围。 实验整体设计：分离15.5日龄小鼠胚胎的胫骨，并将其划分为三个独立区域；从每个节段中提取总RNA，同窝胚胎的各节段RNA进行混合。 实验整体设计：将样本与Affymetrix MOE 430 2.0小鼠基因芯片进行杂交以开展分析；每个区域设置4次独立重复实验。 实验整体设计：时间点数量：1个 实验整体设计：处理组数量：0组 实验整体设计：样本数量：每个区域设置4次生物学重复 实验整体设计：所用基因芯片：MOE 430 2.0 实验整体设计：组织来源：胫骨 实验整体设计：实验物种：E15.5小鼠 实验整体设计：样本类型：总RNA