Department of Spine Surgery, The Tenth Affiliated Hospital of Southern Medical University (Dongguan People's Hospital), Dongguan 523059, China; Department of Orthopaedics, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, Orthopedic Hospital of Guangdong Province, The Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China.
Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.
Cell Signal. 2024 Aug;120:111222. doi: 10.1016/j.cellsig.2024.111222. Epub 2024 May 8.
Bone development involves the rapid proliferation and differentiation of osteogenic lineage cells, which makes accurate chromosomal segregation crucial for ensuring cell proliferation and maintaining chromosomal stability. However, the mechanism underlying the maintenance of chromosome stability during the rapid proliferation and differentiation of Prx1-expressing limb bud mesenchymal cells into osteoblastic precursor cells remains unexplored.
A transgenic mouse model of RanGAP1 knockout of limb and head mesenchymal progenitor cells was constructed to explore the impact of RanGAP1 deletion on bone development by histomorphology and immunostaining. Subsequently, G-banding karyotyping analysis and immunofluorescence staining were used to examine the effects of RanGAP1 deficiency on chromosome instability. Finally, the effects of RanGAP1 deficiency on chromothripsis and bone development signaling pathways were elucidated by whole-genome sequencing, RNA-sequencing, and qPCR.
The ablation of RanGAP1 in limb and head mesenchymal progenitor cells expressing Prx1 in mice resulted in embryonic lethality, severe cartilage and bone dysplasia, and complete loss of cranial vault formation. Moreover, RanGAP1 loss inhibited chondrogenic or osteogenic differentiation of mesenchymal stem cells (MSCs). Most importantly, we found that RanGAP1 loss in limb bud mesenchymal cells triggered missegregation of chromosomes, resulting in chromothripsis of chromosomes 1q and 14q, further inhibiting the expression of key genes involved in multiple bone development signaling pathways such as WNT, Hedgehog, TGF-β/BMP, and PI3K/AKT in the chromothripsis regions, ultimately disrupting skeletal development.
Our results establish RanGAP1 as a critical regulator of bone development, as it supports this process by preserving chromosome stability in Prx1-expressing limb bud mesenchymal cells.
骨骼发育涉及成骨谱系细胞的快速增殖和分化，这使得准确的染色体分离对于确保细胞增殖和维持染色体稳定性至关重要。然而，Prx1 表达的肢芽间充质细胞快速增殖和分化为成骨前体细胞过程中维持染色体稳定性的机制尚不清楚。
构建了 RanGAP1 敲除肢头部间充质祖细胞的转基因小鼠模型，通过组织形态学和免疫染色来探讨 RanGAP1 缺失对骨骼发育的影响。随后，采用 G 带核型分析和免疫荧光染色来检测 RanGAP1 缺失对染色体不稳定性的影响。最后，通过全基因组测序、RNA-seq 和 qPCR 阐明了 RanGAP1 缺失对染色体重排和骨骼发育信号通路的影响。
在 Prx1 表达的小鼠肢头部间充质祖细胞中敲除 RanGAP1 导致胚胎致死、严重的软骨和骨骼发育不良以及颅顶完全缺失。此外，RanGAP1 缺失抑制间充质干细胞（MSCs）的软骨或成骨分化。最重要的是，我们发现肢芽间充质细胞中 RanGAP1 的缺失会导致染色体的错误分离，导致染色体 1q 和 14q 的染色体重排，进一步抑制染色体重排区域中涉及多个骨骼发育信号通路的关键基因的表达，如 WNT、Hedgehog、TGF-β/BMP 和 PI3K/AKT，最终破坏骨骼发育。
我们的研究结果确立了 RanGAP1 作为骨骼发育的关键调节因子的地位，因为它通过在 Prx1 表达的肢芽间充质细胞中维持染色体稳定性来支持这一过程。
