Single-cell analysis identifies a key role for Hhip in murine coronal suture development

Craniofacial development depends on formation and maintenance of sutures between bones of the skull. In sutures, growth occurs at osteogenic fronts along the edge of each bone and suture mesenchyme separates adjacent bones. We performed single-cell RNA-seq analysis of the embryonic, murine coronal suture. Seven populations at E16.5 and nine at E18.5 comprised the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog (HH) signaling, marked a mesenchymal population distinct from other neurocranial sutures. At E18.5, Hhip-/- coronal osteogenic fronts were closely apposed and HH signaling was increased throughout the depleted suture mesenchyme compared to WT, demonstrating that Hhip is required for normal coronal suture development. Tracing of the neonatal Hhip-expressing population showed that descendant cells persisted in the coronal suture and contributed to calvarial bone growth. Our transcriptomic approach provides a rich resource for insight into normal and abnormal development. Laser capture microdissection (LCM) was performed on fresh frozen sections of the coronal (C), lambdoid (L), sagittal (S), and frontal (IF) sutures of C57BL/6J mice at embryonic stages E16.5 and E18.5. Five biological replicates of the suture mesenchyme (SM) and osteogenic front regions were obtained from each suture. RNA was isolated from each LCM sample, converted to RNA-Seq libraries, and sequenced on the Illumina HiSeq 2500 platform. For each suture and time point, pairwise comparisons were then performed for each osteogenic front region compared to suture mesenchyme to identify differentially expressed genes between front and mesenchymal regions.颅面发育依赖于颅骨骨缝的形成与维持。在骨缝中，生长发生于每块骨边缘的成骨前沿（osteogenic fronts），骨缝间充质（suture mesenchyme）分隔相邻骨骼。我们对胚胎期小鼠的冠状骨缝开展了单细胞RNA测序（single-cell RNA-seq）分析。在E16.5时期存在7种细胞群，E18.5时期存在9种细胞群，涵盖骨缝间充质、成骨细胞及相关细胞群。Hhip作为音猬因子（hedgehog, HH）信号通路的抑制剂，其表达标记了一类区别于其他神经颅骨缝的间充质细胞群。在E18.5时期，与野生型（WT）小鼠相比，Hhip基因敲除（Hhip-/-）小鼠的冠状骨缝成骨前沿紧密贴合，且整个缺失的骨缝间充质区域内HH信号通路活性显著升高，这表明Hhip对于冠状骨缝的正常发育是必需的。对新生小鼠Hhip阳性细胞群的谱系追踪显示，其子代细胞持续存在于冠状骨缝中，并参与颅盖骨的生长发育。本研究的转录组学分析策略为阐明正常与异常颅面发育机制提供了丰富的研究资源。我们对C57BL/6J小鼠胚胎期E16.5和E18.5的冠状（C）、人字（lambdoid, L）、矢状（sagittal, S）及额间（IF）骨缝的冰冻新鲜切片进行了激光捕获显微切割（laser capture microdissection, LCM）。从每种骨缝中分别获取了5份骨缝间充质（suture mesenchyme, SM）与成骨前沿区域的生物学重复样本。从每份LCM样本中提取RNA，构建RNA测序文库，并在Illumina HiSeq 2500测序平台上完成测序。针对每种骨缝与每个时间点，我们将成骨前沿区域与骨缝间充质区域进行两两比较，以鉴定两类区域之间的差异表达基因。