Qi Shuqun, Wang Yating, Wei Xiaoxi, Xie Di, Mohsen Rawan, Hsieh Yuan-Lynn, Mishina Yuji, Liu Fei
Department of Biologic and Materials Sciences & Prosthodontics, University of Michigan School of Dentistry, 1011 N University Ave, Ann Arbor, MI, 48109, USA.
State Key Laboratory of Oral Diseases and National Clinical Research Center for Oral Diseases and Other Research Platforms and Department of Orthodontics and Pediatric Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China.
Transgenic Res. 2022 Jun;31(3):399-411. doi: 10.1007/s11248-022-00308-8. Epub 2022 May 8.
The cranial base synchondroses are growth centers that drive cranial and upper facial growth. The intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) are two major synchondroses located in the middle of the cranial base and are maintained at early developmental stages to sustain cranial base elongation. In this study, we report unexpected premature ossification of ISS and SOS when Cre recombinase is activated in a chondrocyte-specific manner. We used a Cre transgenic line expressing Aggrecan enhancer-driven, Tetracycline-inducible Cre (ATC), of which expression is controlled by a Col2a1 promoter. Neonatal doxycycline injection or doxycycline diet fed to breeders was used to activate Cre recombinase. The premature ossification of ISS and/or SOS led to a reduction in cranial base length and subsequently a dome-shaped skull. Furthermore, the mice carrying either heterozygous or homozygous conditional deletion of Tsc1 or Fip200 using ATC mice developed similar craniofacial abnormalities, indicating that Cre activity itself but not conditional deletion of Tsc1 or Fip200 gene, is the major contributor of this phenotype. In contrast, the Col2a1-Cre mice carrying Cre expression in both perichondrium and chondrocytes and the mice carrying the conditional deletion of Tsc1 or Fip200 using Col2a1-Cre did not manifest the same skull abnormalities. In addition to the defective craniofacial bone development, our data also showed that the Cre activation in chondrocytes significantly compromised bone acquisition in femur. Our data calls for the consideration of the potential in vivo adverse effects caused by Cre expression in chondrocytes and reinforcement of the importance of including Cre-containing controls to facilitate accurate phenotype interpretation in transgenic research.
颅底软骨结合是驱动颅骨和上颌面部生长的生长中心。蝶骨间软骨结合（ISS）和蝶枕软骨结合（SOS）是位于颅底中部的两个主要软骨结合，在发育早期得以维持，以支持颅底延长。在本研究中，我们报告了以软骨细胞特异性方式激活Cre重组酶时，ISS和SOS出现意外的过早骨化。我们使用了一种表达由聚集蛋白聚糖增强子驱动、四环素诱导型Cre（ATC）的Cre转基因品系，其表达由Col2a1启动子控制。通过给新生小鼠注射强力霉素或给种鼠喂食含强力霉素的饲料来激活Cre重组酶。ISS和/或SOS的过早骨化导致颅底长度缩短，进而形成圆顶状颅骨。此外，使用ATC小鼠对Tsc1或Fip200进行杂合或纯合条件性缺失的小鼠出现了类似的颅面异常，这表明是Cre活性本身而非Tsc1或Fip200基因的条件性缺失是该表型的主要促成因素。相比之下，在软骨膜和软骨细胞中均表达Cre的Col2a1-Cre小鼠，以及使用Col2a1-Cre对Tsc1或Fip200进行条件性缺失的小鼠并未表现出相同的颅骨异常。除了颅面骨发育缺陷外，我们的数据还表明，软骨细胞中的Cre激活显著损害了股骨的骨量获取。我们的数据呼吁考虑软骨细胞中Cre表达在体内可能产生的不良影响，并强调在转基因研究中纳入含Cre对照以促进准确表型解释的重要性。
