Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
Dev Biol. 2022 Mar;483:76-88. doi: 10.1016/j.ydbio.2021.12.016. Epub 2021 Dec 29.
The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.
小鸡肢芽具有可塑性，可以在早期发育阶段切除一部分间质质量以形成孔后，重建正常的骨骼模式。为了了解孔闭合和重新建立正常肢体轴以重建正常肢体骨骼的详细信息，我们专注于孔修复和肢体恢复过程中的细胞和分子变化。我们从鸡后肢芽的内侧区域（阶段 23）切除了一个块状的间质细胞，并观察了以下形态发生。切除后 15 小时孔已闭合，随后肢芽形态恢复，48 小时后软骨模式基本恢复。间质细胞的谱系分析表明，孔闭合处的孔前缘和后缘的细胞相邻，而近端和远端的细胞则不相邻。为了研究孔修复过程中的细胞极性，我们分析了高尔基器相对于细胞核在细胞内的定位。我们发现，在前缘和后缘的细胞中，高尔基器倾向于朝向孔，而在正常肢芽或孔近端和远端边缘的相同位置的细胞中则没有。在操纵的肢芽中，与对照侧相比，细胞增殖频率保持不变。Tbx3 表达通常局限于肢芽的前缘和后缘，但在肢芽重建过程中，Tbx3 表达暂时向内侧扩展，然后恢复正常模式，Tbx3 阴性细胞重新出现在肢芽的内侧区域。因此，间质孔闭合和肢体重建主要由孔前缘和后缘的细胞介导。这些结果表明，沿前后轴调整细胞特性对于恢复肢体损伤和重建正常骨骼模式至关重要。
