Suture-specific regulation of coronal and lambdoid suture patency by PTHLH and HHIP activity in mice

Craniofacial development depends on the formation of fibrous joints, or sutures, between skull bones. Premature fusion of sutures, or craniosynostosis, is a common human pathology. Ectopic Hedgehog (HH) signaling is one cause of craniosynostosis. Hhip encodes an inhibitor of HH ligands, and we previously identified coronal suture dysgenesis in embryonic Hhip-/- mice, in which suture mesenchyme was depleted between closely opposed but unfused osteogenic fronts at E18.5. Here, we report that the lambdoid suture fuses in Hhip-/- mice by E18.5. RNA-seq analysis of the Hhip-/- coronal and lambdoid sutures show that HH target gene expression, including Pthlh, is upregulated. Paradoxically, expression of Ihh is downregulated. We hypothesized that PTHLH, a negative regulator of Ihh expression, may reduce HH signaling to promote coronal suture patency and prevent fusion of the Hhip-/- coronal suture. We generated Hhip-/-;Pthlh-/- embryos and found that coronal sutures are fusing by E18.5. Our results reveal a previously undescribed role for Pthlh in suture development and demonstrate suture-specific roles for HH inhibitors in maintaining suture patency. To comprehensively identify and compare the transcriptional consequences of loss of Hhip within coronal and lambdoid sutures, we performed RNA-seq analysis of Hhip-/- and WT sutures at E18.5 in a murine C57BL/6, Swiss-Webster, 129 background. Up to four mice were used in each group.颅面发育依赖于颅骨之间纤维关节（即颅缝）的形成。颅缝过早融合，或称颅缝早闭，是一种常见的人类病理状态。异位Hedgehog（HH）信号通路异常是颅缝早闭的诱因之一。Hhip基因编码HH配体的抑制剂，我们此前曾在胚胎期Hhip-/-小鼠中发现冠状颅缝发育不良：在胚胎发育第18.5天（E18.5）时，紧密相邻但未融合的成骨前沿之间的颅缝间充质出现耗竭。本研究发现，Hhip-/-小鼠的人字颅缝在E18.5时发生融合。对Hhip-/-小鼠冠状及人字颅缝的RNA测序（RNA-seq）分析显示，包括Pthlh在内的HH靶基因表达均上调，但出乎意料的是，Ihh的表达却出现下调。我们据此提出假说：作为Ihh表达负调控因子的PTHLH，或许可通过减弱HH信号通路，维持冠状颅缝的开放状态，从而阻止Hhip-/-小鼠冠状颅缝的过早融合。我们构建了Hhip-/-;Pthlh-/-双敲除胚胎，观察到其冠状颅缝在E18.5时发生融合。本研究揭示了Pthlh在颅缝发育中此前未被报道的功能，并证实HH信号通路抑制剂在维持颅缝开放状态中具有颅缝特异性的作用。为全面鉴定并对比Hhip基因缺失在冠状及人字颅缝中造成的转录组变化，我们对处于C57BL/6、Swiss-Webster及129小鼠品系背景下、E18.5时的Hhip-/-及野生型（WT）颅缝样本进行了RNA-seq分析，每组实验最多使用4只小鼠。