Neural crest cells (NCC) arise from the dorsal margin of the neural plate border and comprise a unique cell population that migrates to and creates the craniofacial region. Although factors including Shh, Fgf8, and bone morphogenetic proteins have been shown to regulate these biological events, the role of parathyroid hormone 1 receptor (Pth1r) has been less studied. We generated an NCC-specific mouse model for Pth1r and researched gene expression, function, and interaction focusing on nasal cartilage framework and midfacial development. Wnt1-Cre;Pth1r fl/ fl; Tomato fl/+ mice had perinatal lethality, but we observed short snout and jaws, tongue protrusion, reduced NCC-derived cranial length, increased mineralization in nasal septum and hyoid bones, and less bone mineralization at interfrontal suture in mutants at E18.5. Importantly, the mutant nasal septum and turbinate cartilage histologically revealed gradual, premature accelerated hypertrophic differentiation. We then studied the underlying molecular mechanisms by performing RNA seq analysis and unexpectedly found that expression of Ihh and related signaling molecules was enhanced in mutant nasomaxillary tissues. To see if Pth1r and Ihh signaling are associated, we generated a Wnt1-Cre; Ihh fl/ fl; Pth1r fl/ fl; Tomato fl/+ (DKO) mouse and compared the phenotypes to those of each single knockout mouse: Wnt1-Cre; Ihh fl/ fl; Pth1r fl/+; Tomato fl/+ (Ihh-CKO) and Wnt1-Cre;Ihh fl/+; Pth1r fl/ fl; Tomato fl/+ (Pth1r-CKO). Ihh-CKO mice displayed a milder effect. Of note, the excessive hypertrophic conversion of the nasal cartilage framework observed in Pth1r-CKO was somewhat rescued DKO embryos. Further, a half cAMP responsive element and the 4 similar sequences containing 2 mismatches were identified from the promoter to the first intron in Ihh gene. Gli1-Cre ERT2; Pth1r fl/ fl; Tomato fl/+, a Pth1r-deficient model targeted in hedgehog responsive cells, demonstrated the enlarged hypertrophic layer and significantly more Tomato-positive chondrocytes accumulated in the nasal septum and ethmoidal endochondral ossification. Collectively, the data suggest a relevant Pth1r/Ihh interaction. Our findings obtained from novel mouse models for Pth1r signaling illuminate previously unknown aspects in craniofacial biology and development.

中文翻译：

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神经嵴细胞中的 Pth1r 调节鼻软骨分化

神经嵴细胞 (NCC) 产生于神经板边界的背侧边缘，并包含迁移到并创建颅面区域的独特细胞群。尽管 Shh、Fgf8 和骨形态发生蛋白等因子已被证明可以调节这些生物事件，但甲状旁腺激素 1 受体 (Pth1r) 的作用研究较少。我们为 Pth1r 生成了 NCC 特异性小鼠模型，并研究了基因表达、功能和相互作用，重点关注鼻软骨框架和中面部发育。Wnt1-Cre;Pth1r佛罗里达州/佛罗里达州; 番茄佛罗里达州/+小鼠具有围产期致死率，但我们观察到 E18.5 突变体的口鼻部和下颌短、舌头突出、NCC 源性颅骨长度缩短、鼻中隔和舌骨矿化增加以及额间缝骨矿化减少。重要的是，突变的鼻中隔和鼻甲软骨在组织学上显示出逐渐的、过早的加速肥厚分化。然后，我们通过RNA seq分析研究了潜在的分子机制，意外地发现突变鼻上颌组织中Ihh和相关信号分子的表达增强。为了查看 Pth1r 和 Ihh 信号传导是否相关，我们生成了 Wnt1-Cre；呃佛罗里达州/佛罗里达州; Pth1r佛罗里达州/佛罗里达州; 番茄佛罗里达州/+(DKO) 小鼠，并将表型与每只单一敲除小鼠的表型进行比较：Wnt1-Cre；呃佛罗里达州/佛罗里达州; Pth1r佛罗里达州/+; 番茄佛罗里达州/+(Ihh-CKO) 和 Wnt1-Cre;Ihh佛罗里达州/+; Pth1r佛罗里达州/佛罗里达州; 番茄佛罗里达州/+(Pth1r-CKO)。Ihh-CKO 小鼠表现出较温和的效果。值得注意的是，在 Pth1r-CKO 中观察到的鼻软骨框架过度肥大转化在一定程度上是挽救了 DKO 胚胎。此外，从Ihh基因的启动子到第一个内含子，鉴定出半个cAMP响应元件和含有2个错配的4个相似序列。Gli1-CreERT2; Pth1r佛罗里达州/佛罗里达州; 番茄佛罗里达州/+，一种针对刺猬反应细胞的 Pth1r 缺陷模型，证明了鼻中隔和筛骨软骨内骨化中肥厚层增大，并且番茄阳性软骨细胞明显增多。总的来说，数据表明存在相关的 Pth1r/Ihh 相互作用。我们从 Pth1r 信号传导的新型小鼠模型中获得的研究结果阐明了颅面生物学和发育中以前未知的方面。