Departamento de Anatomía y Biología Celular and Instituto de Formación e Investigación Marqués de Valdecilla, Universidad de Cantabria, Santander, Spain.
PLoS One. 2011;6(9):e24546. doi: 10.1371/journal.pone.0024546. Epub 2011 Sep 13.
The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The "pre-condensation stage", occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the "condensation stage" is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the "pre-cartilage period", precedes the formation of molecularly identifiable cartilage by 2-3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo.
鉴定参与软骨形成的基因对于改进基于细胞的软骨再生疗法至关重要。在这里，我们开发了一种合适的实验模型，通过在发育中的胚胎中诱导异位指来鉴定体内早期的软骨生成事件。在该模型中，在植入 TGFβ珠后仅 12 小时，在没有增加细胞增殖的情况下，软骨就在未分化的指间中胚层中形成，并且在发育过程中成为结构和形态正常的指。系统的定量 PCR 表达分析以及其他实验方法使我们能够在软骨形成之前建立 3 个连续的时期。“预凝聚阶段”发生在治疗的前 3 小时内，其特征是结缔组织特征转录因子（如 Sox9 和 Scleraxis）和分泌因子（如 Activin A 和细胞外基质蛋白 CCN-1 和 CCN-2）的激活以及半乳糖凝集素 CG-8 的下调。接下来，“凝聚阶段”的特征是 Smad 1/5/8 BMP 信号的强烈激活和细胞外基质成分的表达增加。在此期间，CCN 细胞外基质蛋白促进细胞外基质和细胞黏附成分的表达。第三个时期，称为“预软骨期”，在分子上可识别的软骨形成前提前 2-3 小时，其特征是 Sox9 基因表达的增强，以及其他促软骨生成转录因子（如 Hif1a）的刺激。总之，这项工作确立了软骨分化前的前软骨生成基因调控的时间层次，并为体内指导这一过程的第一步的分泌因子和细胞骨架调节剂的相对作用提供了新的见解。
