Gene expression in embryonic intervertebral disc and vertebrae

Very little is known about how intervertebral disc (IVD) is formed or maintained. Members of the TGF-beta superfamily are secreted signaling proteins that regulate many aspects of development including cellular differentiation. We recently showed that deletion of Tgfbr2 in Col2a expressing tissue results in alterations in development of IVD annulus fibrosus. The results suggested TGF-beta has an important role in regulating development of the axial skeleton, however, the mechanistic basis of TGF-beta action in these specialized joints is not known. One of the hurdles to understanding development of IVD is a lack of known markers. To identify genes that are enriched in the developing IVD and to begin to understand the mechanism of TGF-beta action in IVD development, we undertook a global analysis of gene expression comparing gene expression profiles in developing vertebrae and IVD. We also compared expression profiles in tissues from wild type and Tgfbr2 mutant mice. Lists of IVD and vertebrae enriched genes were generated. Expression patterns for several genes were verified either through in situ hybridization or literature/ database searches resulting in a list of genes that can be used as markers of IVD. Cluster analysis using genes listed under the Gene Ontology terms multicellular organism development and pattern specification indicated that mutant IVD more closely resembled vertebrae than wild type IVD. We propose TGF-beta has two functions in IVD development: 1) to prevent chondrocyte differentiation in the presumptive IVD and 2) to promote differentiation of annulus fibrosus from sclerotome. We have identified genes that are enriched in the IVD and regulated by TGF-beta that warrant further investigation as regulators of IVD development. Thirteen samples were analyzed. This includes three biological replicates of laser captured IVD from E13.5 day control mice, three biological replicates of laser captured vertebrae from the same E13.5 day control mice, three biological relicates of laser captured vertebrae from E13.5 day Col2aCre;Tgfbr2lox/lox mice, and four biological replicates of laser captured IVD from E13.5 day Col2aCre;Tgfbr2lox/lox mice.目前人们对椎间盘（intervertebral disc, IVD）的形成与维持机制知之甚少。转化生长因子β（transforming growth factor-beta, TGF-beta）超家族成员是一类分泌型信号蛋白，可调控包括细胞分化在内的诸多发育过程。我们此前的研究证实，在表达Col2a的组织中敲除转化生长因子β受体II型（Tgfbr2）会导致椎间盘纤维环发育异常。该结果提示TGF-beta在轴向骨骼发育中发挥重要作用，但TGF-beta在这类特化关节中的作用机制仍不明确。理解椎间盘发育的一大障碍在于缺乏公认的特异性标记物。为鉴定发育中椎间盘富集的基因并初步阐明TGF-beta在椎间盘发育中的作用机制，我们开展了全基因组基因表达分析，对比了发育中的椎骨与椎间盘的基因表达谱；同时还对比了野生型与Tgfbr2突变小鼠组织的基因表达谱。最终我们得到了椎间盘与椎骨富集基因列表，并通过原位杂交（in situ hybridization）或文献/数据库验证了多个基因的表达模式，由此得到了可作为椎间盘标记物的基因列表。基于基因本体（Gene Ontology）术语“多细胞生物发育”与“模式特化”的基因进行聚类分析后发现，突变型椎间盘的表达谱更接近椎骨，而非野生型椎间盘。我们推测TGF-beta在椎间盘发育中具备两项功能：1）抑制预设椎间盘区域的软骨细胞分化；2）促进生骨节向纤维环分化。我们已鉴定出一批在椎间盘内富集且受TGF-beta调控的基因，这些基因有望作为椎间盘发育的调控因子开展后续研究。本研究共分析了13个样本，具体包括：来自胚胎13.5天（E13.5）对照小鼠的激光捕获椎间盘样本3个生物学重复、同批次对照小鼠的激光捕获椎骨样本3个生物学重复、来自胚胎13.5天（E13.5）Col2aCre;Tgfbr2lox/lox小鼠的激光捕获椎骨样本3个生物学重复，以及同批次突变小鼠的激光捕获椎间盘样本4个生物学重复。