Developmental and molecular cell biological study of the mechanisms of limb differentiation

我们已经报道了所谓的“程序性细胞死亡”（PCD）在趾间组织的胚胎小鼠肢体是细胞凋亡与DNA片段化和区域异质性和时间顺序的细胞死亡在发展中的肢体图案中起着至关重要的作用。然而，没有鉴定出经历细胞分裂的细胞，也没有鉴定出在手指分离期间ECM如何被破坏的过程。我们研究了小鼠胎儿趾间组织中微血管网络的重塑，发现细胞死亡发生在PECAM 1/C 031阳性内皮细胞中。整体原位杂交和北方印迹分析显示，明胶酶/MMP 2在血管重塑发生的区域高表达。在槌状趾突变（Hm/Hm）小鼠胎儿的趾间蹼持续存在的肢体中，既没有观察到细胞凋亡，也没有观察到趾间组织中微血管丛的退化正常发生。我们的研究提供了第一个证据，即细胞凋亡发生在趾间微血管丛的内皮细胞中，并且血管重塑和明胶酶/MMP 2表达可能在哺乳动物趾的分离中起重要作用。接下来，为了分析发育肢体中软骨形成模式的形成机制，我们利用肢体间充质细胞的微团培养系统，并在体外检查软骨形成模式的形成。肢体软骨形成的周期性在微团培养中得以保留。我们开发了一种新的方法来自动测量模式的周期性，并证明反应扩散模型是最合理的解释周期性软骨形成模式的形成。我们还证实了TGFβ2在软骨形成模式中作为“激活剂”样分子，并可能负责细胞分选现象。

We have reported that so-called "programmed cell death" (PCD) in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation and that the regional heterogeneity and temporal sequence of cell death in the developing limb play essential roles in patterning the limb. However, the cells which undergo apoptosiss were not identified nor was the process of how the ECM is disrupted during digit separation. We examined the remodeling of the microvascular network in the interdigital tissue of mouse fetuses and found that cell death occurs in PECAM1/C031-positive endothelial cells. Whole-mount in situ hybridization and Northern blot analysis revealed that gelatinase/MMP2 was highly expressed in the areas where vascular remodeling was occurring. In the limb of Hammertoe mutant (Hm/Hm) mouse fetuses in which interdigital webbing persists, neither apoptosis nor the regression of microvascular plexus in the interdigital tissue were observed to occur normally. Our study has provided the first evidence that apoptotic cell death occurs in endothelial cells of the interdigital microvascular plexus and that vascular remodeling and gelatinase/MMP2 expression may play essential roles in the separation of mammalian digits.Next, to analyze the mechanisms of chondrogenic pattern formation in the developing limb, we utilized a micromass culture system of limb mesenchymal cells, and examined the chondrogenic pattern formation in vitro. The periodic nature of limb chondrogenesis was retained in micromass culture. We developed a novel method to automatically measure the periodicity of the pattern and demonstrated that the reaction-diffusion model is the most plausible for explaining the periodic chondrogenic pattern formation. We also demonstrated that TGFβ2 acts as an "activator"-like molecule in chondrogenic pattern formation and is possibly responsible for the cell sorting phenomenon.