Lee K K, Li F C, Yung W T, Kung J L, Ng J L, Cheah K S
Department of Anatomy, Chinese University of Hong Kong, Shatin.
Dev Dyn. 1994 Dec;201(4):297-309. doi: 10.1002/aja.1002010402.
We have cultured tissues isolated from the interdigital zones (IDZ) of the mouse footplate in the presence of the digits, ectoderm, and all-trans retinoic acid. The objective was to understand how these various factors influence the developmental fate of the interdigital tissues. Neutral red staining showed that these tissues normally differentiate by dying between day 12.5-14.5. However, if they were isolated from the footplate between day 12.5-13.5 (when cell death is not overtly obvious in the IDZ) and maintained in organ culture, these tissues would develop into cartilage and soft connective tissues. In culture, chondrogenesis is initiated very rapidly in the interdigital explants as revealed by in situ hybridization with riboprobes specific for type IIA and IIB procollagen mRNAs. The ability of interdigital tissues to form cartilage is not attributed to factors present in the serum of the culture medium as this phenomenon is also observed in serumless cultures. We have found that if all-trans retinoic acid, at concentrations of 10-50 ng/ml culture medium, were added to the explants it could inhibit chondrogenesis and promote cell death. Moreover, in some of the cultures, a single digit was left attached to the interdigital tissue. This also dramatically reduced the incidence of chondrogenesis. We have tried to determine whether the digits and ectoderm can produce a diffusible factor that can prevent cartilage from developing by culturing day 12.5 interdigital tissues in ectoderm and digit conditioned media. The ectoderm conditioned medium had no effects on interdigital growth or chondrogenesis. In contrast, the size of interdigital explants cultured in the presence of digit conditioned medium was shown to be significantly smaller than the control. These explants also produced a smaller quantity of cartilage as revealed by Alcian blue binding assay. In sum, our results showed that the fate of the interdigital tissues are not fully determined until after day 13.5. These tissues have the potentials to form cartilage and soft connective tissues. We tentatively propose that these interdigital tissues do not normally realize their histogenetic potentials because of the antichondrogenic influence of the digits and retinoic acid.
我们在有趾、外胚层和全反式维甲酸存在的情况下，培养了从小鼠足板指间区域（IDZ）分离出的组织。目的是了解这些不同因素如何影响指间组织的发育命运。中性红染色显示，这些组织通常在12.5 - 14.5天之间通过死亡进行分化。然而，如果在12.5 - 13.5天之间将它们从足板分离出来（此时IDZ中的细胞死亡并不明显）并维持在器官培养中，这些组织会发育成软骨和软结缔组织。在培养中，通过与IIA型和IIB型前胶原mRNA特异性的核糖探针进行原位杂交显示，指间外植体中软骨形成迅速启动。指间组织形成软骨的能力并非归因于培养基血清中存在的因素，因为在无血清培养中也观察到了这种现象。我们发现，如果向外植体中添加浓度为10 - 50 ng/ml培养基的全反式维甲酸，它可以抑制软骨形成并促进细胞死亡。此外，在一些培养物中，单个趾与指间组织相连。这也显著降低了软骨形成的发生率。我们试图通过在12.5天的指间组织在外胚层和趾条件培养基中培养来确定趾和外胚层是否能产生一种可扩散的因子来阻止软骨发育。外胚层条件培养基对指间生长或软骨形成没有影响。相比之下，在趾条件培养基存在下培养的指间外植体的大小明显小于对照。如阿尔新蓝结合试验所示，这些外植体产生的软骨量也较少。总之，我们的结果表明，指间组织的命运直到13.5天后才完全确定。这些组织有形成软骨和软结缔组织的潜力。我们初步提出，由于趾和维甲酸的抗软骨形成影响，这些指间组织通常无法实现其组织发生潜力。
