Department of Wound Repair and Rehabilitation Medicine, State Key Laboratory of Trauma, Burns and Combined Injury, Daping Hospital, Army Medical University, Chongqing 400042, China.
Research Center for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Theranostics. 2020 May 30;10(16):7111-7130. doi: 10.7150/thno.45286. eCollection 2020.
CATSHL syndrome, characterized by camptodactyly, tall stature and hearing loss, is caused by loss-of-function mutations of fibroblast growth factor receptors 3 (FGFR3) gene. Most manifestations of patients with CATSHL syndrome start to develop in the embryonic stage, such as skeletal overgrowth, craniofacial abnormalities, however, the pathogenesis of these phenotypes especially the early maldevelopment remains incompletely understood. Furthermore, there are no effective therapeutic targets for this skeleton dysplasia. We generated knockout zebrafish by CRISPR/Cas9 technology to study the developmental mechanisms and therapeutic targets of CATSHL syndrome. Several zebrafish transgenic lines labeling osteoblasts and chondrocytes, and live Alizarin red staining were used to analyze the dynamical skeleton development in mutants. Western blotting, whole mount in situ hybridization, Edu labeling based cell proliferation assay and Wnt/β-catenin signaling antagonist were used to explore the potential mechanisms and therapeutic targets. We found that mutant zebrafish, staring from early development stage, showed craniofacial bone malformation with microcephaly and delayed closure of cranial sutures, chondroma-like lesion and abnormal development of auditory sensory organs, partially resembling the clinical manifestations of patients with CATSHL syndrome. Further studies showed that regulates the patterning and shaping of pharyngeal arches and the timely ossification of craniofacial skeleton. The abnormal development of pharyngeal arch cartilage is related to the augmented hypertrophy and disordered arrangement of chondrocytes, while decreased proliferation, differentiation and mineralization of osteoblasts may be involved in the delayed maturation of skull bones. Furthermore, we revealed that deficiency of leads to enhanced IHH signaling and up-regulated canonical Wnt/β-catenin signaling, and pharmacological inhibition of Wnt/β-catenin could partially alleviate the phenotypes of mutants. Our study further reveals some novel phenotypes and underlying developmental mechanism of CATSHL syndrome, which deepens our understanding of the pathogenesis of CATSHL and the role of in skeleton development. Our findings provide evidence that modulation of Wnt/β-catenin activity could be a potential therapy for CATSHL syndrome and related skeleton diseases.
CATSHL 综合征的特征为并指畸形、身材高大和听力损失，由成纤维细胞生长因子受体 3 (FGFR3)基因突变引起。大多数 CATSHL 综合征患者的临床表现始于胚胎期，如骨骼过度生长、颅面异常，但这些表型特别是早期发育不良的发病机制仍不完全清楚。此外，对于这种骨骼发育不良还没有有效的治疗靶点。
我们利用 CRISPR/Cas9 技术生成了 基因敲除斑马鱼，以研究 CATSHL 综合征的发育机制和治疗靶点。使用几种标记成骨细胞和软骨细胞的斑马鱼转基因系，并进行活体茜素红染色，以分析 突变体的动态骨骼发育。利用 Western blot、整体原位杂交、Edu 标记细胞增殖检测和 Wnt/β-catenin 信号通路拮抗剂，探讨潜在的机制和治疗靶点。
我们发现，从早期发育阶段开始， 突变体斑马鱼表现出颅面骨畸形，包括小头畸形和颅缝闭合延迟、软骨瘤样病变和听觉感觉器官的异常发育，部分类似于 CATSHL 综合征患者的临床表现。进一步的研究表明， 调节咽弓的模式形成和塑形以及颅面骨骼的适时骨化。咽弓软骨的异常发育与软骨细胞的过度肥大和排列紊乱有关，而成骨细胞的增殖、分化和矿化减少可能与颅骨成熟延迟有关。此外，我们揭示了 缺失导致 IHH 信号增强和经典 Wnt/β-catenin 信号上调，而 Wnt/β-catenin 的药理学抑制可部分缓解 突变体的表型。
我们的研究进一步揭示了 CATSHL 综合征的一些新表型和潜在的发育机制，加深了我们对 CATSHL 发病机制和 基因在骨骼发育中作用的认识。我们的发现为调节 Wnt/β-catenin 活性作为 CATSHL 综合征及相关骨骼疾病的潜在治疗方法提供了证据。
