Skeletal Stem Cell Powering Cranial Suture Fate: An Answer to Craniosynostosis (bulk_RNA-seq)

Purpose: The cranial suture is a fibrous joint, and similar to the growth plates of the skeletal long bone, they serve as the major centers of calvarial vault morphogenesis. Our group's identification of a skeletal stem cell isolated from the mouse tibial growth plate prompted us to investigate whether these skeletal stem cells are also resident in the mouse cranial sutures and if they govern postnatal suture patency or fusion. Results: We preformed a spatio-temporal profiling of the mouse cranial sutures by flow cytometry, demonstrating a significant decrease in the temporal representation of skeletal stem cells in fusing versus patent sutures. Moreover, canonical Wnt signaling has a significant impact on skeletal stem cells proliferation and thus representation within the suture, dictating fate: fusion or patency. Breeding an Axin2+/-LacZ mouse, with enhanced activation of canonical Wnt signaling to a Twist1+/- mouse, harboring a coronal craniosynostosis enriched the skeletal stem cell pool in coronal sutures, thereby preventing Twist1+/- craniosynostosis. Conclusions: Our findings suggest an imbalance and/or decrease in resident skeletal stem cells within the cranial sutures gives rise to craniosynostosis, however, restoring this representation by enriching skeletal stem cells within the suture can maintain patency. Overall design: Total RNA profiles of SSCs isolated from post-natal day 3 posterior frontal (PF), sagittal (Sag), coronal (Cor) sutures and the growth plate (GP) of the tibial long bone from CD-1, wild-type mice were generated by sequencing using Illumina Hi-Seq 4000.研究目的：颅缝（cranial suture）是一类纤维连接结构，与骨骼长骨的生长板（growth plate）相似，是颅盖骨形态发生的核心位点。本团队此前从小鼠胫骨生长板中分离得到骨骼干细胞（skeletal stem cell, SSC），由此开展本研究，旨在探究此类骨骼干细胞是否也驻留于小鼠颅缝中，以及其是否调控出生后颅缝的通畅状态或融合进程。 研究结果：本研究通过流式细胞术（flow cytometry）对小鼠颅缝进行了时空谱分析（spatio-temporal profiling），结果显示，相较于保持通畅的颅缝，发生融合的颅缝中骨骼干细胞的时序占比显著降低。此外，经典Wnt信号通路（canonical Wnt signaling）对骨骼干细胞的增殖及颅缝内的占比具有显著调控作用，并决定颅缝的最终命运：融合或维持通畅。将经典Wnt信号通路激活增强的Axin2+/-LacZ小鼠与携带冠状颅缝早闭（craniosynostosis）表型的Twist1+/-小鼠进行杂交后，冠状颅缝内的骨骼干细胞库得到显著富集，从而逆转了Twist1+/-小鼠的冠状颅缝早闭表型。 研究结论：本研究结果表明，颅缝内驻留骨骼干细胞的数量失衡和/或减少会引发颅缝早闭；而通过富集颅缝内的骨骼干细胞以恢复其正常占比，则可维持颅缝的通畅状态。 实验整体设计：本研究从CD-1品系野生型小鼠的出生后第3天的后额（PF）、矢状（Sag）、冠状（Cor）颅缝以及胫骨长骨生长板（GP）中分离骨骼干细胞，随后采用Illumina Hi-Seq 4000测序技术生成其总RNA转录组图谱（total RNA profiles）。