Molecularbiological study of craniofacial dysmorphology in transgenic mice bearing Apert type mutant Fgfr2 gene

为了验证软骨是突变FGFR2信号传导的另一个靶点的假设，我们在软骨细胞中选择性地产生了表达携带Apert综合征型突变（FGFR2 IIIc^<P253R>）的FGFR2 IIIc转基因小鼠。这些小鼠明显表现出颅骨畸形，包括颅底软骨缝合线过早融合（联合软骨），伴有圆顶颅骨和大开口的异位缝合线，上颌发育不全和前颅底缩短。作为突出的颅骨畸形的可能原因，我们假设FGF配体的特定表达模式可以在颅软骨中产生异常的FGFR2信号。激光显微切片(LMD)/实时荧光定量PCR（R-PCR）和器官培养系统的基因表达分析结果表明，Fgfi2 IIIc^<P253R>的表达获得了自分泌调节，与FGF2和FGF10配体在颅底软骨中的特定分布模式一致。这些结果表明，颅底软骨的异常发育是Apert综合征优先颅面畸形的另一个基本机制。为了阐明FGFR2信号激活的生物学效应，我们使用LMD/R-PCR系统分析了软骨细胞系细胞的分化标志物。与组织学发现一致，LMD/R-PCR数据表明Cbfa1、Ihh和MMP-13参与了软骨细胞的肥厚分化。综上所述，颅软骨中FGFR2信号的异常激活可导致软骨细胞终末分化加速，从而导致颅面畸形伴颅联合软骨过早融合。

To test a hypothesis that the cartilage is another target for the mutant FGFR2 signaling, we generated transgenic mice expressing Fgfr2 IIIc bearing Apert syndrome type mutation (Fgfr2 IIIc^<P253R>) selectively in chondrocytes. These mice markedly manifested deformities of the cranium including premature fusion of cartilaginous sutures in the cranial base(synchondroses), which accompanied by domed skull with wide opened metopic suture, maxillary hypoplasia, and shortening of anterior cranial base. As a likely cause of the prominent cranial deformities, we hypothesized specific expression patterns of FGF ligands that could produce aberrant FGFR2 signaling in cranial cartilage. The results of gene expression analyses by Lasermicrodisection(LMD)/Real time PCR(R-PCR) and organ culture system with FGFs stimulation demonstrated the acquirement of edopic autocrine regulation by Fgfi2 IIIc^<P253R> expression in concert with the specific distribution pattern of FGF2 and FGF10 ligand in cranial base cartilage. These results propose importance role of abnormal development of the cranial base cartilage as another basic mechanism of the preferential craniofacial dysmorphologies in Apert syndrome. To clarify the biological effect of activation of FGFR2 signaling, we used the LMD/R-PCR system to analyze the differentiation markers for chondrocyte lineage cells. Consistent with histological findings, LMD/R-PCR data have suggested activation of the molecules, Cbfa1, Ihh and MMP-13,which are involved in hypertrophic differentiation of the chondrocyte. Taken together, aberrant activation of FGFR2 signaling in cranial cartilage could result acceleration of chondrocyte terminal differentiation, consequently, resulting the craniofacial dysmorphology with premature fusion of cranial synchondroses.